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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.

In order to increase the rate of kojic production using Aspergillus oryzae , the initial glucose and Polypepton concentrations were optimized by the Box-Wilson method. A kojic acid production rate of 4.44 g/ l ·d was obtained when the initial glucose and Polypepton concentrations were 148.0 g/ l and 4.8 g/ l , respectively, at a K L a value of 164.7 h −1 .

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.

A 5.1 kb plasmid, pCDR1, which conferred cadmium resistance on Escherichia coli C600, was obtained during the cloning of the cadmium resistance gene from pGU100 harbored by cadmium-resistant Pseudomonas putida GAM-1, pCDR1 was constructed with pBR322 as vector and an about 800 bp inserted fragment. The nucleotide sequence revealed that the inserted DNA fragment was IS1. The cadmium resistance of this transformant was maintained even though the plasmid was cured. Southern analysis showed that the mobility of one band was changed, indicating that a chromosomal rearrangement occurred in the cadmium-resistant strain.

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.

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.

A cadmium-sensitive mutant strain was isolated from a Cd2+-tolerant bacterium, Pseudomonas aeruginosa G-1 strain by mutagenesis with N-methyl-N′-nitro-N-mtrosoguanidine. The Cd2+-sensitive mutant strain was about 8-times as sensitive to Cd2+ and 2-times as sensitive to Hg2+, Cr2+ and achromycin, and not sensitive to Cu2+, as the parent strain.Vacuole-like substances were observed in the parent strain by transmission electron microscope in the presence of Cd2+, but not in the absence of Cd2+. The mutant strain took up 3.3-times the amount of Cd2+ into cells when compared with the parent strain. Disc electrophoretic patterns showed a characteristic protein band in the parent strain at the stationary phase, but the corresponding protein band was present in the mutant strain at the late exponential phase.

A base-specific ribonuclease (RNase) Lu (EC 3.1.27.5) was isolated and purified from Aspergillus niger in a yield of 23% by means of acetone precipitation, column chromatography on Duolite A-2, DEAE-cellulose, and 2'(3')-aminohexyl-5'-ATP-Sepharose 4B. The enzyme was shown to be homogeneous by disc-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 43000 by both gel filtration and SDS-gel electrophoresis. The isoelectric point of the enzyme was determined to be 3.75 by isoelectric focusing. The enzyme had a base specificity: it released only 3'-UMP from yeast RNA or poly(U) and, in addition, small amounts of 3'-CMP from poly(C).

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.

In Osaka city, solid waste have been disposed in a pond at a sea‐based solid waste disposal site. The leachate is treated by the combined process of a facultative pond and an aerated lagoon. We examined the microflara of the combined process and clarified the relationship between leachate loading and microbial activities. BODs in the aerated lagoon was stably below 20 mgl−1 year round. The dominant genera in the aerated lagoon were Alcaligenes, Microcaccus, Maraxella, Aeramonas and Acinetabacter during the lower leachate loading period. There were no pathogenic bacteria of the genera Salmonella, Shigella and Vibrio in the aerated lagoon.

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.

A mercuric chloride-tolerant bacterium was isolated from activated sludge and the bacterium was identified as Pseudomonas oleovorans.

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.

L-Arabinose ketol-isomerase (EC 5. 3. 1. 4) was extracted from the L-arabinose-grown cells of Clostridium acetobutylicum and partially purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and the gel filtration on Sephadex G-200. Optimum pH and temperature for the activity of the enzyme were 7.5 and 37°C, respectively. The enzyme was inhibited by EDTA, Na-pyrophosphate and KCN, and its activity was restored by the addition of divalent metal ions such as Mn2+ and Co2+. The Michaelis-Menten's constants for L-arabinose, Mn2+ and Co2+ were 1.08×10-2M, 0.83×10-4M and 0.38×10-4M, respectively. The equilibrium constant (K=ribulose/arabinose) was 3.14 (37◊C, pH 7.5). The thermodynamic quantities, ΔH, ΔG (37°C), ΔS (37°C) and E were +7354cal/mole, -710.7cal/mole, +26.0cal/(deg•mole) and +7471cal/mole, respectively.

Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose. With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH4H2PO4 and MgSO4·7H2O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days.

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.

イチジク緑葉成分の中プソラレン,精油及び精油のフェノール部分の微生物に対する抗菌抗黴性を検討した.プソラレンは72時間後に於て細菌類には5千倍以下, Asp. oryzae 1万倍, Hans. anomala 10万倍でその発育を阻止した.精油及び精油のフェノール部分はプソラレンよりも抗菌性が劣っており,精油は72時間後に於て各微生物に対し5千倍以下の抗菌性を示す程度であり,精油のフェノール部分はB. subtilis, Staph. aureus及びPseud. fluorescensに対し2千倍, Asp. oryzae及びHans. anomalaに対し1千倍の稀釈濃度でその発育を阻止した.

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.

Cadmium-resistant pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl/sub 2/ as high as 7 mM, was isolated from soil in a rice paddy. This bacterium harbored a DNA plasmid of about 52 kilobases. The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance. A cadmium-resistant transformant of E. coli C600 contained a plasmid corresponding to that seen in P. putida GAM-1. The transformant did not take up cadmium as well as P. putida GAM-1 did. This is the first report on the mechanism of plasmid-determined Cd/sup 2 +/ resistance in gram-negative bacteria.