Hirofumi Shinoyama

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

ABSTRACT Iodide (I−)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive 125I−. When they were grown in a liquid medium containing 0.1 μM 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 μM 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 × 103, respectively. In the presence of much higher concentrations of iodide (1 μM 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 μM and 0.55 pmol min−1 mg of dry cells−1, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO3−), was not transported.

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

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of β-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%).

Dissimilatory Iodate Reduction by Marine Pseudomonas sp. Strain SCT

ABSTRACT Bacterial iodate (IO3−) 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 μM 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 μM 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 characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Abstract Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.

Hydrogen Peroxide-Dependent Uptake of Iodine by Marine Flavobacteriaceae Bacterium Strain C-21

ABSTRACT 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.

Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus

Abstract A thermostable catalase from the culture broth of Thermoascus aurantiacus grown on ethanol was purified as an electrophoretically and isoelectrophoretically homogeneous protein. The molecular weights of the native enzyme and its subunit were estimated to be approximately 330,000 and 75,000 by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was proven to be a homo-tetrameric hemecatalase with one iron atom per subunit and about 11% carbohydrate. The pI was 4.5. The enzyme was stable up to 80°C and over a wide range of pH from 5 to 13. The optimum pH was in the range of 6 to 10 and the optimum temperature was 70°C. The K m and K cat of the enzyme for hydrogen peroxide were 4.8 × 10 −2 M and 1.07 × 10 5 s −1 , respectively. The enzyme was strongly inhibited by potassium cyanide and sodium azide.

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°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.

Radiotracer Experiments on Biological Volatilization of Organic Iodine from Coastal Seawaters

Biological volatilization of iodine from seawaters was studied using a radiotracer technique. Seawater samples were incubated aerobically in serum bottles with radioactive iodide tracer (125I), and volatile organic and inorganic iodine were collected with activated charcoal and silver wool trap, respectively. Iodine was volatilized mainly as organic iodine, and inorganic iodine volatilization was not observed. Influence of light intensity on the volatilization was determined, but no significant differences were observed under light (70,000 lux) and dark conditions. The effect of the chemical form of iodine on the volatilization was determined, and the results suggested that volatilization preferentially occurs from iodide (I−) but not from iodate (IO3 −). Volatilization did not occur when the samples were autoclaved or filtered through a 0.22-μm pore size membrane filter. Incubation of the samples with antibiotics caused decreased volatilization. Conversely, enhanced volatilization was observed when the samples were incubated with yeast extract. Fifty-nine marine bacterial strains were then randomly isolated from marine environments, and their iodine-volatilizing capacities were determined. Among these, 19 strains exhibited significant capacities for volatilizing iodine. 16S ribosomal RNA gene comparisons indicated that these bacteria are members of Proteobacteria (α and γ subdivisions) and Cytophaga-Flexibacter-Bacteroides group. One of the strains, strain C-19, volatilized 1 to 2% of total iodine during cultivation, and the gaseous organic iodine was identified as methyl iodide (CH3I). These results suggest that organic iodine volatilization from seawaters occurs biologically, and that marine bacteria participate in the process. Considering that volatile organic iodine emitted from the oceans causes atmospheric ozone destruction, biological iodine volatilization from seawater is of great importance. Our results also contribute to prediction of movement and diffusion of long-lived radioactive iodine (129I) in the environment.

Enzymatic Synthesis of 4-Hydroxyphenyl β-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 β-D-oligoxylosides, β-(Xyl)n-HQ (n=1–4), by one step reaction. The obtained β-(Xyl)n-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC50 values of β-Xyl-HQ, β-(Xyl)2-HQ, β-(Xyl)3-HQ, and β-(Xyl)4-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. β-(Xyl)4-HQ showed 35-fold more potent inhibitory activity than β-arbutin (4-hydroxyphenyl β-D-glucopyranoside), of which the IC50 value was measured to be 6.3 mM. Kinetic analysis revealed that β-(Xyl)2-HQ, β-(Xyl)3-HQ, and β-(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 β-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 β-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 β-oligoxylosides.