Enzymatic synthesis of 4-hydroxyphenyl beta-D-oligoxylosides and their notable tyrosinase inhibitory activity.

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)(E), was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl beta-D-oligoxylosides, beta-(Xyl)(n)-HQ (n=1-4), by one step reaction. The obtained beta-(Xyl)(n)-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC(50) values of beta-Xyl-HQ, beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. beta-(Xyl)(4)-HQ showed 35-fold more potent inhibitory activity than beta-arbutin (4-hydroxyphenyl beta-D-glucopyranoside), of which the IC(50) value was measured to be 6.3 mM. Kinetic analysis revealed that beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ competitively inhibited the enzyme, and the corresponding K(i) values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

Characterization of a novel polyphenol-specific oligoxyloside transfer reaction by a family 11 xylanase from Bacillus sp. KT12.

A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl beta-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4-11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a beta-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl beta-oligoxylosides.

A Direct Method to Isolate DNA from Phyllosphere Microbial Communities without Disrupting Leaf Tissues.

We developed a method for direct DNA isolation from phyllosphere microbial communities, designated Direct-DIP. This method comprises DNA extraction from non-shredded leaves with benzyl chloride, and DNA purification by gel filtration. Scanning electron microscopy showed that epiphytic microorganisms were completely removed from the leaf surface after benzyl chloride treatment, while microstructures of the leaf were not damaged. Clear DGGE profiles were obtained regardless of the plant species. Shannon diversity indices of DGGE profiles by Direct-DIP were higher than those by a conventional method. Our findings suggest that Direct-DIP is a rapid, simple, and cost-effective method of extracting DNA from phyllosphere microbial communities.

Production of fructooligosaccharides by crude enzyme preparations of beta-fructofuranosidase from Aureobasidium pullulans.

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of beta-fructofuranosidases (FFases) obtained from sucrose-cultured cells of Aureobasidium pullulans DSM 2404. When the preparation mainly consisted of FFase I, that has high transfructosylating activity, the FOS yield was 62%. When the reaction was carried out with additional commercial glucose isomerase (GI) at an activity ratio of FFase and GI of 1:2, the maximum FOS yield reached 69%. This value was higher than those obtained previously using other Aureobasidium spp. (53-59%).

Hydrogen peroxide-dependent uptake of iodine by marine Flavobacteriaceae bacterium strain C-21.

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I-). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, 125I-. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.

Dissimilatory iodate reduction by marine Pseudomonas sp. strain SCT.

Bacterial iodate (IO(3)(-)) reduction is poorly understood largely due to the limited number of available isolates as well as the paucity of information about key enzymes involved in the reaction. In this study, an iodate-reducing bacterium, designated strain SCT, was newly isolated from marine sediment slurry. SCT is phylogenetically closely related to the denitrifying bacterium Pseudomonas stutzeri and reduced 200 microM iodate to iodide (I(-)) within 12 h in an anaerobic culture containing 10 mM nitrate. The strain did not reduce iodate under the aerobic conditions. An anaerobic washed cell suspension of SCT reduced iodate when the cells were pregrown anaerobically with 10 mM nitrate and 200 microM iodate. However, cells pregrown without iodate did not reduce it. The cells in the former category showed methyl viologen-dependent iodate reductase activity (0.31 U mg(-1)), which was located predominantly in the periplasmic space. Furthermore, SCT was capable of anaerobic growth with 3 mM iodate as the sole electron acceptor, and the cells showed enhanced activity with respect to iodate reductase (2.46 U mg(-1)). These results suggest that SCT is a dissimilatory iodate-reducing bacterium and that its iodate reductase is induced by iodate under anaerobic growth conditions.

Purification and some properties of beta-fructofuranosidase I formed by Aureobasidium pullulans DSM 2404.

beta-Fructofuranosidase I (FFase I) formed by Aureobasidium pullulans DSM 2404 was purified. The enzyme had a molecular weight of about 430 kDa, was not affected by various metal ions and showed high transfructosylating activity. The yield of fructooligosaccharides production using purified FFase I was 62%.

16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique.

We have developed an analytical technique for the 16S rRNA gene that comprises whole-genome amplification and the polymerase chain reaction (PCR)-minigel-single-strand conformation polymorphism technique (WGA-SSCP). Under optimal conditions, SSCP bands could be detected when genomic DNA from bacteria of interest comprised 0.5% or more of the specimen. This method will be effective for the identification of nonculturable bacteria in a microbial community.

Multiple beta-fructofuranosidases by Aureobasidium pullulans DSM2404 and their roles in fructooligosaccharide production.

At least five types of beta-fructofuranosidases (FFases I, II, III, IV and V) were found in the cell wall of Aureobasidium pullulans DSM2404 grown in a sucrose medium. The fungus first catalyzed the transfructosylation of sucrose, and produced fructooligosaccharide (FOS) and glucose in the culture. FOS was then consumed together with glucose, and finally fructose was produced. In the FOS-producing period, the fungus expressed FFase I as a dominant FFase. However, in the FOS-degrading period, the levels of FFases II, III, IV and V increased. The ratios of transfructosylating activity to hydrolyzing activity by FFases I-V were 14.3, 12.1, 11.7, 1.28 and 8.11, respectively. When glucose was used as a carbon source, only FFase I showed significant activity. On the other hand, the activities of all five FFases were detected when FOS or fructose was used as a carbon source. These results suggested that the expression of FFase I was not repressed by glucose, but those of FFases II-V were strongly inhibited in the presence of glucose. It is considered that FFase I plays a key role in FOS production by this fungus, whereas FFase IV may function as a FOS-degrading enzyme with its strong hydrolyzing activity.

Inducible production of alcohol oxidase and catalase in a pectin medium by Thermoascus aurantiacus IFO 31693.

Thermoascus aurantiacus showed the best growth on medium containing pectin as a carbon source. The enzyme involved in the production of catalase in the fungus was alcohol oxidase. Formaldehyde dehydrogenase and formate dehydrogenase, in addition to alcohol oxidase and catalase, were detected in the cells grown on pectin. Alcohol oxidase was alkali resistant (pH 7 to 11), and was comparatively heat stable (55 degrees C).

Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693.

Intracellular and extracellular alcohol oxidases (AO int and AO ext) were purified from the liquid and solid cultures of a thermophilic fungus, Thermoascus aurantiacus NBRC 31693, as electrophoretically and isoelectrophoretically homogeneous proteins, respectively. Both enzymes contained a flavin adenine dinucleotide (FAD) cofactor and were stained with Schiff's reagent. The molecular weight of AO int was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AO ext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AO int and AO ext were 5.88 and 6.08, respectively. AO int and AO ext were stable up to 60 degrees C and 55 degrees C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AO int oxidized short straight-chain alcohols (K(m) for methanol, 13.5 mM and K(m) for ethanol, 15.8 mM). On the other hand, AO ext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (K(m) for methanol, 0.5 mM and K(m) for ethanol, 10.2 mM).

Generation and characterization of reduced virulence Fusarium oxysporum f. sp. lycopersici mutants through plasmid-vector insertion.

Pathogenicity-impaired mutants, B02 and H15, of Fusarium oxysporum f. sp. lycorpersici (FOL) were obtained using restriction enzyme-mediated integration. Disease severities of Fusarium wilt caused by these mutants were significantly reduced, and their disease development rates were correlated with their colonization rates in tomato vessels. Both B02 and H15 produced significantly smaller amounts of extracellular proteins as well as fusaric acid than the wild-type. Southern blot analyses suggested that B02 and H15 likely contain a single and three copies of transformation vector, respectively. These mutants may thus be useful in isolating genes involved in pathogenicity of FOL.

Isolation of iodide-oxidizing bacteria from iodide-rich natural gas brines and seawaters.

Iodide-oxidizing bacteria (IOB), which oxidize iodide (I-) to molecular iodine (I2), were isolated from iodide-rich (63 microM to 1.2 mM) natural gas brine waters collected from several locations. Agar media containing iodide and starch were prepared, and brine waters were spread directly on the media. The IOB, which appeared as purple colonies, were obtained from 28 of the 44 brine waters. The population sizes of IOB in the brines were 10(2) to 10(5) colony-forming units (CFU) mL(-1). However, IOB were not detected in natural seawaters and terrestrial soils (fewer than 10 CFU mL(-1) and 10(2) CFU g wet weight of soils(-1), respectively). Interestingly, after the enrichment with 1 mM iodide, IOB were found in 6 of the 8 seawaters with population sizes of 10(3) to 10(5) CFU mL(-1). 16S rDNA sequencing and phylogenetic analyses showed that the IOB strains are divided into two groups within the alpha-subclass of the Proteobacteria. One of the groups was phylogenetically most closely related to Roseovarius tolerans with sequence similarities between 94% and 98%. The other group was most closely related to Rhodothalassium salexigens, although the sequence similarities were relatively low (89% to 91%). The iodide-oxidizing reaction by IOB was mediated by an extracellular enzyme protein that requires oxygen. Radiotracer experiments showed that IOB produce not only I2 but also volatile organic iodine, which were identified as diiodomethane (CH2I2) and chloroiodomethane (CH2ClI). These results indicate that at least two types of IOB are distributed in the environment, and that they are preferentially isolated in environments in which iodide levels are very high. It is possible that IOB oxidize iodide in the natural environment, and they could significantly contribute to the biogeochemical cycling of iodine.

Active transport and accumulation of iodide by newly isolated marine bacteria.

Iodide (I(-))-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive (125)I(-). When they were grown in a liquid medium containing 0.1 microM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 microM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 +/- 3.6 (mean +/- standard deviation) pmol of iodide per mg of dry cells and 5.5 x 10(3), respectively. In the presence of much higher concentrations of iodide (1 microM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 muM and 0.55 pmol min(-1) mg of dry cells(-1), respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO(3)(-)), was not transported.

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7.

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.

Microbial participation in iodine volatilization from soils.

The roles of microorganisms in iodine volatilization from soils were studied. Soils were incubated with iodide ion (I-), and volatile organic iodine species were determined with a gas chromatograph. Iodine was emitted mainly as methyl iodide (CH3I), and CH3I emission was sometimes enhanced by the addition of glucose. Soils were then incubated with a radioactive iodine tracer (125I), and radioiodine emitted from soils was determined. The emission of iodine was enhanced in the presence of yeast extract but was inhibited by autoclaving of soils. The addition of streptomycin and tetracycline, antibiotics that inhibit bacterial growth, strongly inhibited iodine emission, while a fungal inhibitor cycloheximide caused little effect. Forty bacterial strains were randomly isolated from soils, and their capacities for volatilizing iodine were determined. Among these, 14 strains volatilized significant amounts of iodine when they were cultivated with iodide ion. Phylogenetic analysis based on 16S ribosomal DNA sequences showed thatthese bacteria are widely distributed through the bacterial domain. Our results suggest that iodine in soils is methylated and volatilized as CH3I by the action of soil bacteria and that iodine-volatilizing bacteria are ubiquitous in soil environments. The pathway of iodine volatilization by soil bacteria should be important for understanding the biogeochemical cycling of iodine as well as for the assessment of long-lived radioactive iodine (129I) in the environment.

Some properties of glycine aminotransferase purified from Rhodopseudomonas palustris No. 7 concerning extracellular porphyrin production.

Glycine aminotransferase (EC 2.6.1.4; GlyAT) was presumed to be an enzyme concerning the supply of glycine for the extracellular porphyrin production by Rhodopseudomonas palustris No. 7. GlyAT was purified from strain No. 7 as an electrophoretically homogenous protein. The enzyme was a monomer protein with the molecular weight of about 42,000. From the absorption spectrum of the enzyme (350 nm, 410 nm), it was indicated that the enzyme had pyridoxal phosphate as a prosthetic group. The enzyme showed high substrate specificity for glutamate as an amino group donor. Apparent Kms for glutamate and glyoxylate were 6.20 mM and 3.75 mM, respectively. The Vmax and Kcat for glutamate were 66.8 mumol/min/mg protein and 46.8 s-1, respectively. The Vmax and Kcat for glyoxylate were 68.8 mumol/min/mg protein and 48.2 s-1. The optimum temperature and pH were 40-45 degrees C and 7.0-7.5, respectively. The enzyme activity lowered to about 50% in the presence of 15 mM ammonium chloride.

Relationship between conidial enzymes and germination of the apple blue mold, Penicillium expansum.

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.