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A xylanase gene, xynA, which encodes xylanase I of Aeromonas caviae ME-1, was cloned in Escherichia coli. xynA gene has a 633 bp open reading frame (encoding 211 amino acid residues) which includes a signal peptide (28 amino acid residues). E. coli JM109 transformant produced 0.7 U/ml xylanase, about 80% of which was secreted into the culture medium as mature enzyme. The deduced amino acid sequence shows 79.3, 78.9, and 47.1% identity to family G xylanases of Bacillus subtilis, Bacillus pumilus and Bacillus circulans, respectively.

A non-woven fabric was successfully applied for immobilization of Candida tropicalis to produce xylitol from d-xylose. Xylitol productivity was enhanced by feeding of d-glucose (50 g/l·d); 87 g xylitol/L was produced after 64 h cultivation. Non-woven fabric could be used five times for fed-batch cultivation.

d -Glucose fed-batch cultivation was studied for effective production of xylitol from d -xylose by Candida tropicalis. When d -xylose was used as a single carbon source, the yeast consumed d -xylose for production of xylitol as well as for growth and cell maintenance resulting in decreased xylitol yield. To improve the xylitol yield, it is necessary to increase the cell mass using another carbon source. We selected d -glucose for the purpose and as a result, a 1.1–1.2 times higher yield and a 1.2–1.3 times higher production rate were obtained by supplementation of d -glucose using column reactors (200 ml scale). In a bench-scale fermentor (3 l scale) experiment, xylitol was produced at up to 104.5 g/l at 32 h cultivation and a yield of 0.82 (g/g d -xylose consumed) and production rate of 3.26 g/l·h were obtained. These values are 1.3 times and 1.2 times higher for yield and production rate, respectively, than those obtained under optimum cultivation conditions for production rate previously reported.

NADPH-dependent d -xylose reductases (XRs) from Candida tropicalis IFO 0618 were purified and characterized. Mono Q HPLC revealed three XR isomers. The K m values of XR1, XR2 and XR3 for d -xylose were 37, 30 and 34 mM, and for NADPH 14, 18 and 9 μM, respectively. NADH did not act as a cofactor. The specificities of the three XRs for several aldoses were essentially the same. Gel filtration and cross-linking analysis showed that both XR1 and XR2 were dimers composed of identical subunits. The pI values of XR1 and XR2 were estimated to be 4.15 and 4.10, respectively. Comparison of the peptide maps of XR1 and XR2 showed that the molecular weights of 8 fragments of lysylendopeptidase-digested XR1 and XR2 were essentially the same as each other. The amino acid composition of each XR was also very similar. The molecular weights of XR1 and XR2 by mass spectra analysis were 36,497.91 and 36,539.68, respectively. The amino acid sequences of two XR1 peptide fragments (Nos. 4 and 5) were highly homologous with those of Pichia stipitis XR and mammalian aldose reductases.

A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37°C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37°C. When soluble birch xylan was used as the substrate, the enzyme had a K m and V max of 2 mg/ml and 182 μmol of xylose equivalent liberated · min -1 · mg of protein -1 , respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither β-xylosidase, α- l -arabinofuranosidase, cellulase, nor β-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.

D-Xylose isomerase was purified to homogeneity from cell-free extracts of Bifidobacterium adolescentis by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and Butyl-Toyopearl. The molecular weight of the purified enzyme was estimated to be 168,000 by gel filtration on TSKgel G-3000SW, and 53,000 on SDS-polyacrylamide gel electrophoresis. The optimum pH was around 7 and the enzyme was stable at pH 7-8. The enzyme required bivalent cations, Mg2+, Co2+, or Mn2+ for the activity, particularly Mn2+ to be best. The enzyme had a pI of 4.3, and the Km for D-xylose was 4 mM. The N-terminal amino acid sequence of the enzyme was not similar to those of D-xylose isomerases from other sources such as Clostridium thermosulfurogenes, Escherichia coli, or Bacillus subtilis.

The effect of culture conditions on xylitol production rate was investigated using Candida tropicalis IFO 0618. From the variance analysis of xylitol production rate, it was found that initial yeast extract concentration was highly significant (99%), while the interaction between D-xylose concentration and aeration rate was significant (95%). These results show the importance of initial yeast extract concentration and of the balance between D-xylose concentration and aeration in the production of xylitol. It was also clearly shown that C. tropicalis needed more yeast extract concentration for efficient xylitol production than for its growth. In order to enhance xylitol production rate, culture conditions were optimized by the Box-Wilson method. In this respect, initial D-xylose concentration, yeast extract concentration, and K(L)a were chosen as the independent factors in 2(3)-factorial experimental design. As the result of experiments, a maximum xylitol production rate of 2.67 g/L . h was obtained when initial D-xylose concentration and yeast extract concentration were 172.0 and 21.0 g/L, respectively, and K(L)a was 451.50 h(-1) by 90% oxygen gas.

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.

Two d -xylose reductase (XR) genes, xyrA and xyrB , of Candida tropicalis IFO 0618 were cloned and sequenced. Each XR gene had an open reading frame consisting of 324 amino acids (972 bp). The deduced amino acid sequences of each XR gene were very similar to each other, except for three amino acids. The molecular weights of the deduced gene products were 36,551 ( xyrA ) and 36,596 ( xyrB ). These genes were 73.0% identical to the Pichia stipitis XR gene.

The purpose of this study is to prepare Worcestershire sauce with higher levels of ethanol and aromatic components by a trickle bed bioreactor. Ethanol productivity in a trickle bed bioreactor was measured with changing circulation rates from 40 to 280 ml/min with a fixed aeration rate (200 ml/min) at 28°C. Compared with the lower circulation rate (40 ml/min), at 210 ml/min of circulation rate or higher, ethanol productivity increased 10.3-fold at 41 h of fermentation.

To investigate the role of hydrogen peroxide in Cr(VI) toxicity in vivo toward bacterial cells, we examined the effect of Cr(VI), hydrogen peroxide, sodium azide, and mannitol on the viability of Escherichia coli. Bacterial cells were incubated for 1 h with shaking in the presence of Cr(VI), hydrogen peroxide, sodium azide as catalase inhibitor, and/or mannitol as radical scavenger. The colony-forming ability and double-strand DNA degradation were examined. The viability assays revealed that Cr(VI) toxicity depended on hydroxyl radicals generated in the reaction involving hydrogen peroxide and chromium. Moreover, incubation of E. coli cells with 10 mM Cr(VI) and 3 mM hydrogen peroxide caused the degradation of double-strand DNA in vivo, which was suppressed by the addition of mannitol. These results indicated that hydroxyl radicals generated in the incubation degraded DNA of E. coli cells, resulting in cell death. In the absence of added hydrogen peroxide, the intracellular concentration of hydrogen peroxide in E. coli was low (below 1 microM). A catalase-defective strain incubated in the absence of added hydrogen peroxide remained fully viable after 1 h but showed decreased viability after prolonged incubation (4-8 h). The addition of mannitol suppressed this decrease, suggesting that hydroxyl radicals may be involved in the expression of Cr(VI) toxicity even without added hydrogen peroxide.

A strain of Byssochlamys sp. produced β-hydroxypropionic acid when grown on media containing high concentrations of acrylic acid. The maximal production of β-hydroxypropionic acid was 4.8% when the initial culture medium contained 7% acrylic acid and 2% glucose, and the initial culture pH was adjusted to 7.0. β-Hydroxypropionic acid production from acrylic acid depended greatly on the pH of the culture medium. Calcium hydroxide was the best neutralizer.

An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme. Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.

When hexavalent chromium (Cr6+) tolerant Pseudomonas ambigua G-l was cultivated in nutrient broth containing 150 ppm Cr6 +, the Cr6+ content of the broth rapidly decreased. The Cr6+ reducing enzyme found in a cell-free extract of P. ambigua G-l required NADH but not NADPH as a hydrogen donor for the reduction of Cr6 +. The specific activities of cell-free extracts of several Cr6+ sensitive mutants derived from P. ambigua G-l showed decreases to one fourth to one tenth of that of P. ambigua G-l. Glucose protected the Cr6+ reducing enzyme against inac-tivation on dialysis.

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from several yeasts were absorbed to blue dextran-Sepharose 4B and eluted with 1 M KCl in high yields. While pyruvate kinase of Candida guilliermodii AHU 4101 was eluted to some extent with phosphoenopyruvate (PEP), ADP or fructose, 1,6-diphosphate (FDP) individually from blue dextran-Sepharose 4B, the combination of FDP with PEP or ADP caused the complete elution of the kinase. Cibacron Blue competitively inhibited the pyruvate kinase with respect to both PEP and ADP at a saturated level of fixed substrate in the presence FDP, an allosteric activator. Further inhibition studies with Cibacron Blue suggest that one molecule of the blue dye binds per active site of the kinase at a saturate level of PEP, but two molecules of the dye bind per active site an unsaturated level of PEP in the presence of FDP.

The chemical components of ribonuclease L (RNase L) from Aspergillus sp. were tested in order to clarify the structure of the enzyme. The molecular weight and the number of the total anuno acid residues of the enzyme were about 4.5 × 104 and 393, respectively. The amino- and carboxyl-terminal amino acids of the enzyme were determined to be glycine and threonine, respectively. RNase L contained about 6% of neutral sugars and an glycopeptide was obtained by digestion with pronase. The isoelectric point of the enzyme was 3.47.

Phosphoglycerate mutases (EC 2.7.5.3) of several lactic acid bacteria were adsorbed to blue dextran-Sepharose 4B and eluted quantitatively with 1 M KC1. Phosphoglycerate mutase of Leuconostoc dextranicum AHU 1078 was eluted with 3-phosphoglycerate, 2-phosphoglycerate or 2,3-bisphosphoglycerate at 1 mM. Furthermore, ATP, ADP, GTP and GDP were also effective for eluting the mutase from the blue dextran-Sepharose column, but AMP and GMP were not. Although NADP(H) eluted the mutase, NAD(H) did not. By single step purification using linear gradient elution of 2,3-bisphosphoglycerate between 0 and 0.5 mM, the Leuconostoc mutase was purified to electrophoretic homogeneity with a 62.5% yield. The difference in spectrum of Cibacron Blue in the presence of the mutase was quite similar to that of the dye in the presence of KC1, but not in the presence of glycerol. The Leuconostoc mutase was inactivated by pyridoxal phosphate. This inactivation was prevented in the presence of blue dextran, as well as 3-phosphoglycerate.

Candida utilis Ribose 5-phosphate ketol isomerase (EC 5.3.1.6) was purified. The enzyme is homogeneous by ultracentrifugal analysis and disc electrophoresis. The isoelectric point of the enzyme is 3.8, Km for R 5-P is 0.108 mM and Kis by 5′-AMP and 6-P-G are 0.16 mM and 0.61 mM, respectively. The thermodynamic parameters, ΔH, ΔG (35°C), ΔS (35°C) and E are 4336 cal/mole, — 5629 cal/mole, 32.15 cal/deg-mole and 6360 cal/mole, respectively. The molecular weight of the enzyme is 183,000 and the enzyme is assumed to consist of three subunits, one with a molecular weight of 75,000 and two identical subunits each with a molecular weight of 54,000.

An Hg2+-sensitive mutant strain was isolated from an Hg2+-tolerant bacterium Pseudomonas oleovorans G-1 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Hg2+-sensitive mutant strain was about 10-times as sensitive to Hg2+ as the parent strain. Moreover, the mutant strain was considerably more sensitive to Cr6+ than the parent strain, but it did not show an appreciable change in sensitivity to Cd2+ and Cu2+. The mutant strain was considerably more sensitive to antibiotics achromycin, chloramphenicol and streptomycin than the parent strain. A more rigid structure was observed in the cell envelope of the mutant strain than the parent strain under transmission electron microscope. Higher amounts of DNA but less protein and RNA were found in the mutant strain compared to the parent strain. Disc electrophoretic patterns showed some differences in protein bands between the parent and mutant strain.

As for the recent research on azoreductase, (i) the reduction mechanism of azoreductase by hepatic cell-free extracts or rat internal contents, (ii) the spectrophotometric determination for the enzyme by rat hepatic microsome, and (iii) the fluorophotometric determination for rat liver cytosol using Methyl Red as a substrate were studied. There is no research on the purification of the enzyme and also on identification of metabolic intermediates using labelled compounds as substrates. In a previous paper, the microorganism, which degraded p-aminoazobenzene into aniline and p-phenylenediamine was isolated from waste waters of dyestuff industries in the Gifu district, Japan, and also reduction, oxidation and acetylation of p-aminoazobenzene were reported. In this paper, the authors describe partial purification procedure of azoreductase, some properties of the azoreductase and identification of degradative intermediates, p-phenylenediamine and aniline, from p-aminoazobenzene using two types of (/sup 14/C)-labelled p-aminoazobenzenes.

The waste waters of dyestuff industries in Gifu district are applied for mixed treatment with municipal sewage by activated sludge. Toxicities of the dye against microorganisms cause degradation of purifying functions in treatment plants. Therefore, when there is a great deal of waste water from dyestuff industries, primary treatment is required with dye-assimilating bacteria in dyestuff industries. In the soil obtained from the drain trench of dyestuff industries in the Hashima district, a few kinds of dye-assimilating bacteria were isolated and identified. To investigate the possibility of primary treatment with these strains, the metabolic pathways of dyestuffs with such bacteria were studied. In the previous study, p-aminoazobenzene was found to be subject to reductive fission of the azo bond and acetylation of the amino group. In a continuation of the study of the previous metabolites, the authors isolated and identified other metabolites, the authors isolated and identified other metabolites, o-aminophenol, m- and p-acetamidophenol, and 3,4-dihydroxyacetanilide, from the culture broth of Pseudomonas cepacia 13NA. The isolation and identification of these metabolites are reported in the present study.

A potassium chromate-tolerant bacterium was isolated from activated sludge, and the bacterium was identified as Pseudomonas ambigua G–1. The bacterium tolerated up to 2000 ppm of Cr6+, 1700 ppm of Cu2+ and 200 ppm of Cd2+, but did not tolerate Hg2+. Chemical analysis indicated 86.5% uptake in the soluble fraction and 13.5% uptake in the insoluble fraction of cells. Chromate uptake distribution in the soluble fraction indicated 28.9% in microsomal fraction and 78.1% in supernatant fraction, Chromate distribution in the insoluble fraction showed 61% in lipid-fraction and 21% in polyphosphate-polysaccharidefraction. Chromate inhibited the syntheses of protein, DNA in soluble fraction and RNA in microsomal fraction.

Phosphoglycerate mutase (EC 2.7.5.3) of Leuconostoc dextranicum AHU 1078 adsorbed onto blue dextran-Sepharose, was easily eluted with a low concentration of pyrophosphate. Pyrophosphate inhibited the mutase competitively with respect to 3-phosphoglycerate, but noncompetitive^ with respect to 2,3-bisphosphoglycerate. Further inhibition studies suggested that two molecules of pyrophosphate bound per active site of the phosphoglycerate mutase. The inactivation of the Leuconostoc mutase by pyridoxal phosphate was prevented by the addition of pyrophosphate. Spectral measurements revealed that the mutase-Cibacron Blue complex in solution was dissociated by the addition of pyrophosphate.

Interspecific protoplast fusion between Candida utilis IAM 4277 and Candida lipolytica OUT 6340 was carried out. A stable fusant, No. 1, was selected from among many fusants which were able to grow on both maltose and /i-hexadecane. Fusant No. 1 seems to be an intermediate between the parents with respect to morphological and physiological characteristics. Further chemotaxonomic studies on the coenzyme Q type, fatty acid composition and enzyme patterns, and antigenic analysis revealed that fusant No. 1 is a hybrid having both a major part of the characteristics of C. lipolytica OUT 6340 and a minor part of those of C. utilis IAM 4277.

5'-Guanosine monophosphate (5'-GMP) was found to leak from Candida lipolytica (IFO, 0746) and its mutant Y and Sm. Mutant Sm obtained from the parent strain by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ultraviolet irradiation was found to leak 5'-GMP at 5 times the amount of the parent strain. The properties of mutant Y and Sm were compared with the parent strain, and differences were present in growth, amount of 5'-GMP leakage and ribonuclease activity.

In order to establish the mechanism of occurrence of sunlight flavour of beer in continuation of the preceding paper, humulone, lupulone, and their analogues and degradation compounds, i.e., cohumulone, adhumulone, 5-acetyl-3-methylfilicinic acid including analogues, tetrahydrohumulone, hexahydrolupulone, humulinic acid and lupuloxinic acid, were tested for the occurrence of sunlight flavour of beer.As a result, among the above-mentioned compounds, humulone, lupulone, cohumulone, adhumulone, tetrahydrohumulone and hexahydrolupulone were found to cause the sunlight flavour, but the other compounds did not cause typical sunlight flavour. This fact shows that some specific structural components seem requisite for the occurrence of sunlight flavour of beer.It was also revealed that isomerization caused by boiling accelerates the occurrence of the sunlight flavour of beer. Finally, the result of the experiment conducted by the gas chromatographic procedure showed that any new component is not detected by the e...

Three synthetic lupulone analogues, i.e., 1-acetyl-3,3,5-tri-allyl-, 1-acetyl-3,3,5-tri-n-propyl-and 1-acetyl-3,3,5-tri-n-butyl-hexadiene- (1,5) -diol- (2,6) -on- (4) did not cause sunlight flavor of beer when they were added to a fermented solution of sucrose and exposed to sunlight.When methanol solutions of humulone and acetolupuphenonen were exposed to sunlight, changes in the UV-absorption spectra were observed. This fact indicates that humulone and acetolupuphenone underwent some photochemical reaction upon exposure to sunlight.Prenyl (γ,γ-dimethylallyl) mercaptan was synthesized, which gave a typical sunlight flavor when it was added, in a trace amount, to a fermented solution of sucrose.

自然界よりPMAおよびMA耐性菌をスクリーニングしたところ,活性汚泥中より耐性の強い菌を分離した.ついで本菌を同定したところ, Pseudomonas ovalisと確認された.また本菌の培養中におけるpH,菌体量, PMAおよびMAの取込み量などの経時的変化,ならびに菌体への取込まれ方について検討した結果, PMAおよびMAは破壊菌体を26,000×g, 15分間で沈降する区分に,大部分取込まれていることがみとめられた.

Xylose isomerase, an enzyme isomerizing xylose to xylulose, was produced adaptively when Candida utilis, a fodder yeast, was grown in a medium containing xylose. This experiment was carried out in order to isolate and purify the enzyme, and to clarify properties of the enzyme. As a result, it was revealed that the enzyme could be solubilized by plasmolysis, and was purified by dialysis, salting out with ammonium sulfate, precipitating with acetone, and adsorbing to calcium phosphate gel. The enzyme requires some divalent metal ions for its action and among them manganese ion is the most effective. The enzyme is inhibited particularly by ethylenediamine-tetraacetic acid and has somewhat thermotolerant character.

In a previous paper, the synthesis of β,-γ-hexenol from the Grignard reagent of pentenylbromide and formaldehyde has been reported.However, it was not established with certainty that the synthetic compound thus obtained was truly β,-γ-hexenol itself. Recently, Harper and Smith traced the procedure outlined in that paper and concluded that the synthetic products were not β,-γ-hexenol and as they mainly contained 2-ethylbut-3-en-l-ol and in addition unsaturated hydrocarbons.Here again, the earlier procedure was repeated on a larger scale.From this, it was recognized that β,-γ-hexenol is not to be gained, but 2-ethylbut-3-en-l-ol and unsaturated hydrocarbons are obtained in accordance with the experiment of Harper and Smith.

l-Arabinose ketol isomerase (EC 5.3.1.4) was extracted from cells of Candida utilis which were grown in sulfite pulp mill waste.The enzyme was partially purified 135-fold by means of fractionation with ammonium sulfate, on DEAE-cellulose and DEAE-Sephadex A-50 column chromatographies and gel filtration on Sephadex G-200.Optimal pH and temperature for the activity of the enzyme were 7.0 and 60°C, respectively. The enzyme reaction was activated by Co2+ and Mn2+, and was inhibited by Na-pyrophosphate.The Michaelis-Menten’s constant for l-arabinose was 1.07×10−1m, and the thermodynamic quantities, ΔH, ΔG (35°C), ΔS (35°C) and E were 8914 cal/mole, −1397 cal/mole, 33.5 cal/deg mole and 9588 cal/mole, respectively.

p-Aminoazobenzene was degraded by Bacillus subtilis to aniline and p-phenylenediamine by reductive fission of an azo bond. The aniline was then acetylated to acetanilide while the p-phenylenediamine underwent 2 successive acetylations to yield p-aminoacetanilide and p-phenylenediacetanilide. In addition, another pathway was found in Bacillus subtilis in which p-aminoazobenzene was metabolised to p-acetamidoazobenzene.

Living Aspergillus niger cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column bioreactors to produce citric acid from sucrose. With the replacement batch bioreactor, increase of citric acid was observed under conditions of higher aeration and of wider surface of immobilized cells. With the continuous bioreactor, the maximum citric acid yield was 39.1 mg/h per 40 g gels. The biocatalyst activity or half-life was 105 days.

The authors earlier isolated a few strains of microbes in sludge from the sewage of an azo dye factory which had assimilability to azo dye. Among them, strain 13NA was identified as Pseudomonas cepacia based on Bergey's Manual and was named Pseudomonas cepacia 13NA. A model experiment for continuous treatment of dye waste was also reported. Some strain 13NA specificities for aminoazobenzenes and reductive and acetylating pathways are described in the present study.