Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and a... more Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and animals and is also an important bacterium for dairy and probiotic supplement production. Therefore, bacteriophages infecting E. faecalis may be useful for phage therapy against multidrug-resistant strains or may threaten industrial fermentation. We isolated a virulent Siphoviridae bacteriophage, BC-611, specifically infecting E. faecalis strain NP-10011 but not infecting other E. faecalis strains or other enterococci. Although the genome sequence of BC-611 resembled that of enterococcal bacteriophage SAP6, BC-611 was marked by its narrow host specificity.
Applied and Environmental Microbiology, Dec 1, 2016
The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus cu... more The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli. Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, -xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited -xylosidase activity toward a chromogenic substrate, p-nitrophenyl--D-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, ␣-L-Araf-(1¡2)-D-Xylp or ␣-L-Araf-(1¡3)-D-Xylp and ␣-L-Araf-(1¡2)-[␣-L-Araf-(1¡3)]--D-Xylp. Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, -xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.
Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes... more Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10-30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.
Applied and Environmental Microbiology, May 1, 1997
Acidiphilium multivorum AIU 301 isolated from acid mineral water had strong arsenic resistance. T... more Acidiphilium multivorum AIU 301 isolated from acid mineral water had strong arsenic resistance. This bacterium harbored a number of plasmids with different molecular sizes. A plasmid of 56 kbp, named pKW301, was isolated from A. multivorum AIU 301. When pKW301 was transferred into Escherichia coli JM109 by electroporation, an E. coli transformant carrying pKW301 exhibited resistance to sodium arsenite, sodium arsenate, and mercuric(II) chloride.
Xylanases as well as cellulases produced by thermophilic bacteria have attracted special interest... more Xylanases as well as cellulases produced by thermophilic bacteria have attracted special interest because of their high potential for the sacchariflcation of cellulosic biomass. Three xylanases, A, B, and C, were purified to homogeneity from a culture filtrate of Clostridium stercorarium and characterized.x) We isolated an anaerobic thermophilic bacterium, strain HX-1, from a compost heap. Strain HX-1 was grown anaerobically at 60°C for 40 hr in GS medium2) with glucose as a carbon source. This bacterium was identified as C. stercorarium^by shape (rod, 0.5 x 4/mi), spore formation, growth at 60°C, GC content (39%), motility, and growth on xylan, xylose, and starch. Anovel xylanase, termed xylanase D, was purified from 1 1 of
An anaerobic two-stage continuous fermentation process with combined thermophilic hydrogenogenic ... more An anaerobic two-stage continuous fermentation process with combined thermophilic hydrogenogenic and methanogenic stages (two-stage fermentation process) was applied to artificial food wastes on a laboratory scale. In this report, organic loading rate (OLR) conditions for hydrogen fermentation were optimized before operating the two-stage fermentation process. The OLR was set at 11.2, 24.3, 35.2, 45.6, 56.1, and 67.3 g-COD cr L-1 day-1 with a temperature of 60 o C, pH5.5 and 5.0% total solids. As a result, approximately 1.8-2.0 mol-H 2 mol-hexose-1 was obtained at the OLR of 11.2-56.1 g-COD cr L-1 day-1. In contrast, it was inferred that the hydrogen yield at the OLR of 67.3 g-COD cr L-1 day-1 decreased because of an increase in lactate concentration in the culture medium. The performance of the two-stage fermentation process was also evaluated over three months. The hydraulic retention time (HRT) of methane fermentation was able to be shortened 5.0 days (under OLR 12.4 g-COD cr L-1 day-1 conditions) when the OLR of hydrogen fermentation was 44.0 g-COD cr L-1 day-1 , and the average gasification efficiency of the two-stage fermentation process was 81% at the time.
The α-galactosidase mel4A (previously called melA) gene of Bacillus halodurans was recombinantly ... more The α-galactosidase mel4A (previously called melA) gene of Bacillus halodurans was recombinantly expressed in Escherichia coli, purified and characterized. The mel4A gene consists of 1305 nucleotides encoding a protein of 434 amino acids with a predicted molecular weight of 49,761. According to its primary structure as deduced from the nucleotide sequence of the gene, Mel4A was assigned to family 4 of glycoside hydrolases. Almost all of the enzyme was produced as inclusion bodies at 37 o C in E. coli. In order to reduce the expression level, cultivation temperature was decreased to 20 o C so that the enzyme could be collected from soluble fraction. Recombinant α-galactosidase Mel4A was purified to homogeneity in a single step using His-binding metal affinity chromatography. B. halodurans Mel4A has the unusual property, i.e., absolutely depending on NAD + and Mn 2+ for activity. Co 2+ and Ni 2+ also activated Mel4A, albeit less efficiently than Mn 2+. In addition, Mel4A activity required reducing condition which met by the addition of dithiothreitol (DTT). In the presence of all cofactors, optimum activity was achieved at 37 o C and pH 7.4. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose but not guar gum, indicating that this enzyme preferred small saccharides to highly polymerized galactomannans. Western immunoblots of intracellular and extracellularproteins of B. halodurans revealed that raffinose induced the expression of intracellular Mel4A of B. halodurans. This bacterium was also able to utilize guar gum as the carbon source, but Western blot analysis indicated that the production of Mel4A was not enhanced by the addition of guar gum.
Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schiz... more Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and dl-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V(infmax) values for dihydroxyacetone were much higher than those for glycerol and dl-glyceraldehyde. On the basis of the K(infm) values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.
Cells of Pseudomonas strain C-l20, cultivated under the conditions in which cells do not floccula... more Cells of Pseudomonas strain C-l20, cultivated under the conditions in which cells do not flocculate naturally, were flocculated with DNA prepared from Escherichia coli, indicating that DNA binding factor was constitutively present on the cell surface. On the other hand, release of DNA into the growth medium was observed accompanying flocculation of cells. The results suggest that release of DNA from cells is an important factor for flocculation. DNA binding activity of cells was abolished by treating cells with proteases, suggesting the DNA binding factor is a proteinaceous component. The effects of salts and 2,4,6-trinitrobenzene sulfonic acid on the cells suggested that amino groups were involved in the DNA binding reaction. The number of DNAs bound per cell was estimated to be about 10 molecules from reconstitution experiments using phage T4 DNA. * Total A660 of a culture was measured after DNase I (20 J.lg/ml) treatment when C-120 cells were harvested. 1) ** Measured when cells were harvested. 1) *** Not determined. Few flocs were observed with the naked eye.
A bacterium belonging to Pseudomonas which was isolated from activated sludge formed floes in gly... more A bacterium belonging to Pseudomonas which was isolated from activated sludge formed floes in glycerol-containing medium. .The floes were deflocculated by deoxyribonuclease treatment in the presence of magnesium ions. Floes were also deflocculated by 2 M NaCl, heating at temperatures higher than 50 a C, and at pH below 1 or above 11. The observations suggest that deoxyribonucleic acid is directly involved in the association of cells and that ionic bonds are responsible for the flocculation of cells.
Nine mutant strains defective in flocculating abilities were derived from the flocculating parent... more Nine mutant strains defective in flocculating abilities were derived from the flocculating parent Pseudomonas strain C-120. Mutations in flocculating ability did not influence the rate and extent of cell growth. Amongthem, six strains which did not form floes at all completely lacked DNAbinding activity. Other mutant strains formed floes to a lesser extent as compared with the parent. Cell envelopes and SDS-extracted envelopes prepared from the parent strain, but not from nonflocculating mutant strains, held DNA-binding activities, indicating that DNA-binding activities of cell envelopes and SDS-extracted envelopes represented that of the wild type cells responsible for flocculation. DNA-binding activities of cell envelopes were abolished by treating with proteases, suggesting that the DNAbinding factor is a proteinaceous component. Susceptibility of DNAbinding activities of SDS-extracted envelopes to 2,4,6-trinitrobenzene sulfonic acid or at high temperatures supported this assumption. Although cell envelopes prepared from the parent cells and mutant cells were subjected to SDS-polyacrylamide gel electrophoresis, no differences in protein composition were observed. Pseudomonas strain C-120, which was isolated from activated sludge, flocculates spontaneously after the late logarithmic growth phase, and floes are rapidly and completely deflocculated by treatment with added deoxyribonuclease (DNase).1) In a previous paper,2) we showed that the component responsible for flocculation was extracted from floes by treatment with 3m guanidine hydrochlo
The Journal of General and Applied Microbiology, 1987
The isoelectric points of serine/alkaline proteinases of various microorganisms reported to date ... more The isoelectric points of serine/alkaline proteinases of various microorganisms reported to date fall in the range of 4.9 to 10.7 (Table 1). Recent work in this laboratory (24) revealed that Aspergillus niger mutant 8, which is a non-acid-productive mutant, produces serine proteinases of an extremely anionic nature (p13.7). This stimulated the authors, who have been concerned with some ecological studies on microbial proteinases (25-27), to determining whether such novel serine proteinases of low p1 values might occur frequently in a group of fungi that, like the above mutant, produce no acid. Twenty-one strains of non-acid-productive fungi (Table 2) were used for the experiment. They were selected from the collection of this laboratory and from unidentified isolates using the surface colony technique of DAvis (28). About a half loopful of spores of each fungus was transferred from the slant to the surface of 1 Ecological Studies on the Proteolytic Enzymes of Microorganisms. Part IV.
Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and a... more Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and animals and is also an important bacterium for dairy and probiotic supplement production. Therefore, bacteriophages infecting E. faecalis may be useful for phage therapy against multidrug-resistant strains or may threaten industrial fermentation. We isolated a virulent Siphoviridae bacteriophage, BC-611, specifically infecting E. faecalis strain NP-10011 but not infecting other E. faecalis strains or other enterococci. Although the genome sequence of BC-611 resembled that of enterococcal bacteriophage SAP6, BC-611 was marked by its narrow host specificity.
Promoter activity of the genes encoding Taka-amylase A and phosphoglycerate kinase of Aspergillus... more Promoter activity of the genes encoding Taka-amylase A and phosphoglycerate kinase of Aspergillus oryzae was analyzed using Escherichia coli ~glucuronidase as a reporter in a shoyu-koji mold A. oryzae KBN616. Assay of the ~ glucuronidase extracted from the mycelia of the transformants grown in the media containing different carbon sources suggests that expression of the Taka-amylase A gene was not induced by starch in A. oryzae KBN616. Analysis of proteins in the culture supernatant ofA. oryzae KBN616 after cultivation in starch medium supports this result. The phosphoglycerate kinase gene of A. oryzae KBN616 was shown to be expressed constitutively in the medium containing glucose and starch.
An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. Ther... more An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. There is a possibility that microorganisms are involved in generation of combustible gas from RDF and this study was aimed at showing the presence of bacteria that can ferment RDF pellets. All RDF samples tested contained a relatively high number of viable bacterial cells, 1:4 Â 10 5 to 3:2 Â 10 6 viable cells/g. These bacteria in the RDF samples fermented them to generate heat and hydrogen gas.
The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 ... more The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula  P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.
A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-... more A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-O-methyl-aD -glucuronosyl-xylotriose (MeGlcAX 3), as a main product from birchwood xylan and accumulated them in the culture under optimum conditions at pH 7.0 and 55°C using 0.75% (w/v) birchwood xylan as a carbon source for 42-72 h. The acidic xylotriose was purified by ethanol precipitation and high-performance liquid chromatography using NH 2 Lichosher® 100 column. The results of electrospray ionization mass spectrometry, mass to charge ratio (m/z) 603.23, confirmed that the purified sample was acidic xylotriose that had benefits and applications in many fields.
The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introd... more The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.
The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The struct... more The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.
The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open readin... more The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl -D-N,N-diacetylchitobioside [4-MU-(GlcNAc) 2 ]. The pH and temperature optima of the enzyme were 6.0 and 45°C, respectively. The K m and V max values for 4-MU-(GlcNAc) 2 were estimated to be 6.3 M and 46 mol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin. Chitinase (EC 3.2.1.14) is a glycosyl hydrolase that catalyzes the degradation of chitin, an insoluble linear -1,4-linked polymer of N-acetylglucosamine. Chitinases are present in a wide range of organisms, including bacteria, insects, viruses, plants, and animals, and play important physiological and ecological roles. On the basis of amino acid sequence homology, chitinases are divided into two unrelated families, families 18 and 19 of glycosyl hydrolases (15). Family 18 includes chitinases from bacteria, fungi, viruses, and animals and chitinases classified in class III or V from plants. On the other hand, family 19 includes almost exclusively plant chitinases classified in classes I, II, and IV but also a bacterial chitinase, Streptomyces griseus HUT 6037 chitinase C (32). To date, various chitinases were isolated from aerobic microorganisms such as Bacillus circulans (1, 55-57), Serratia marcescens (6, 13, 16, 20), an Aeromonas sp. (41, 51-53), an Alteromonas sp. (48), Streptomyces plicatus (37), Streptomyces olivaceoviridis (3, 36, 38), and Janthiobacterium lividum (12). Several genes encoding chitinases were cloned in Escherichia coli and characterized in detail along with their translated products (9, 12, 20, 36-38, 41, 43, 49, 56, 57). From these studies, chitinases were found to comprise two or more discrete domains, while the function of each domain has not yet been elucidated. Watanabe et al. reported that B. circulans ChiA1 has a chitin-binding domain (CBD) and two fibronectin type III (Fn3) domains in addition to a catalytic domain classified in family 18 (59). Deletion analysis indicated that a CBD but not Fn3 domains were responsible for binding of ChiA1 to insoluble chitins and further
Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and a... more Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and animals and is also an important bacterium for dairy and probiotic supplement production. Therefore, bacteriophages infecting E. faecalis may be useful for phage therapy against multidrug-resistant strains or may threaten industrial fermentation. We isolated a virulent Siphoviridae bacteriophage, BC-611, specifically infecting E. faecalis strain NP-10011 but not infecting other E. faecalis strains or other enterococci. Although the genome sequence of BC-611 resembled that of enterococcal bacteriophage SAP6, BC-611 was marked by its narrow host specificity.
Applied and Environmental Microbiology, Dec 1, 2016
The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus cu... more The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli. Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, -xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited -xylosidase activity toward a chromogenic substrate, p-nitrophenyl--D-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, ␣-L-Araf-(1¡2)-D-Xylp or ␣-L-Araf-(1¡3)-D-Xylp and ␣-L-Araf-(1¡2)-[␣-L-Araf-(1¡3)]--D-Xylp. Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, -xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.
Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes... more Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10-30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.
Applied and Environmental Microbiology, May 1, 1997
Acidiphilium multivorum AIU 301 isolated from acid mineral water had strong arsenic resistance. T... more Acidiphilium multivorum AIU 301 isolated from acid mineral water had strong arsenic resistance. This bacterium harbored a number of plasmids with different molecular sizes. A plasmid of 56 kbp, named pKW301, was isolated from A. multivorum AIU 301. When pKW301 was transferred into Escherichia coli JM109 by electroporation, an E. coli transformant carrying pKW301 exhibited resistance to sodium arsenite, sodium arsenate, and mercuric(II) chloride.
Xylanases as well as cellulases produced by thermophilic bacteria have attracted special interest... more Xylanases as well as cellulases produced by thermophilic bacteria have attracted special interest because of their high potential for the sacchariflcation of cellulosic biomass. Three xylanases, A, B, and C, were purified to homogeneity from a culture filtrate of Clostridium stercorarium and characterized.x) We isolated an anaerobic thermophilic bacterium, strain HX-1, from a compost heap. Strain HX-1 was grown anaerobically at 60°C for 40 hr in GS medium2) with glucose as a carbon source. This bacterium was identified as C. stercorarium^by shape (rod, 0.5 x 4/mi), spore formation, growth at 60°C, GC content (39%), motility, and growth on xylan, xylose, and starch. Anovel xylanase, termed xylanase D, was purified from 1 1 of
An anaerobic two-stage continuous fermentation process with combined thermophilic hydrogenogenic ... more An anaerobic two-stage continuous fermentation process with combined thermophilic hydrogenogenic and methanogenic stages (two-stage fermentation process) was applied to artificial food wastes on a laboratory scale. In this report, organic loading rate (OLR) conditions for hydrogen fermentation were optimized before operating the two-stage fermentation process. The OLR was set at 11.2, 24.3, 35.2, 45.6, 56.1, and 67.3 g-COD cr L-1 day-1 with a temperature of 60 o C, pH5.5 and 5.0% total solids. As a result, approximately 1.8-2.0 mol-H 2 mol-hexose-1 was obtained at the OLR of 11.2-56.1 g-COD cr L-1 day-1. In contrast, it was inferred that the hydrogen yield at the OLR of 67.3 g-COD cr L-1 day-1 decreased because of an increase in lactate concentration in the culture medium. The performance of the two-stage fermentation process was also evaluated over three months. The hydraulic retention time (HRT) of methane fermentation was able to be shortened 5.0 days (under OLR 12.4 g-COD cr L-1 day-1 conditions) when the OLR of hydrogen fermentation was 44.0 g-COD cr L-1 day-1 , and the average gasification efficiency of the two-stage fermentation process was 81% at the time.
The α-galactosidase mel4A (previously called melA) gene of Bacillus halodurans was recombinantly ... more The α-galactosidase mel4A (previously called melA) gene of Bacillus halodurans was recombinantly expressed in Escherichia coli, purified and characterized. The mel4A gene consists of 1305 nucleotides encoding a protein of 434 amino acids with a predicted molecular weight of 49,761. According to its primary structure as deduced from the nucleotide sequence of the gene, Mel4A was assigned to family 4 of glycoside hydrolases. Almost all of the enzyme was produced as inclusion bodies at 37 o C in E. coli. In order to reduce the expression level, cultivation temperature was decreased to 20 o C so that the enzyme could be collected from soluble fraction. Recombinant α-galactosidase Mel4A was purified to homogeneity in a single step using His-binding metal affinity chromatography. B. halodurans Mel4A has the unusual property, i.e., absolutely depending on NAD + and Mn 2+ for activity. Co 2+ and Ni 2+ also activated Mel4A, albeit less efficiently than Mn 2+. In addition, Mel4A activity required reducing condition which met by the addition of dithiothreitol (DTT). In the presence of all cofactors, optimum activity was achieved at 37 o C and pH 7.4. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose but not guar gum, indicating that this enzyme preferred small saccharides to highly polymerized galactomannans. Western immunoblots of intracellular and extracellularproteins of B. halodurans revealed that raffinose induced the expression of intracellular Mel4A of B. halodurans. This bacterium was also able to utilize guar gum as the carbon source, but Western blot analysis indicated that the production of Mel4A was not enhanced by the addition of guar gum.
Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schiz... more Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and dl-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V(infmax) values for dihydroxyacetone were much higher than those for glycerol and dl-glyceraldehyde. On the basis of the K(infm) values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.
Cells of Pseudomonas strain C-l20, cultivated under the conditions in which cells do not floccula... more Cells of Pseudomonas strain C-l20, cultivated under the conditions in which cells do not flocculate naturally, were flocculated with DNA prepared from Escherichia coli, indicating that DNA binding factor was constitutively present on the cell surface. On the other hand, release of DNA into the growth medium was observed accompanying flocculation of cells. The results suggest that release of DNA from cells is an important factor for flocculation. DNA binding activity of cells was abolished by treating cells with proteases, suggesting the DNA binding factor is a proteinaceous component. The effects of salts and 2,4,6-trinitrobenzene sulfonic acid on the cells suggested that amino groups were involved in the DNA binding reaction. The number of DNAs bound per cell was estimated to be about 10 molecules from reconstitution experiments using phage T4 DNA. * Total A660 of a culture was measured after DNase I (20 J.lg/ml) treatment when C-120 cells were harvested. 1) ** Measured when cells were harvested. 1) *** Not determined. Few flocs were observed with the naked eye.
A bacterium belonging to Pseudomonas which was isolated from activated sludge formed floes in gly... more A bacterium belonging to Pseudomonas which was isolated from activated sludge formed floes in glycerol-containing medium. .The floes were deflocculated by deoxyribonuclease treatment in the presence of magnesium ions. Floes were also deflocculated by 2 M NaCl, heating at temperatures higher than 50 a C, and at pH below 1 or above 11. The observations suggest that deoxyribonucleic acid is directly involved in the association of cells and that ionic bonds are responsible for the flocculation of cells.
Nine mutant strains defective in flocculating abilities were derived from the flocculating parent... more Nine mutant strains defective in flocculating abilities were derived from the flocculating parent Pseudomonas strain C-120. Mutations in flocculating ability did not influence the rate and extent of cell growth. Amongthem, six strains which did not form floes at all completely lacked DNAbinding activity. Other mutant strains formed floes to a lesser extent as compared with the parent. Cell envelopes and SDS-extracted envelopes prepared from the parent strain, but not from nonflocculating mutant strains, held DNA-binding activities, indicating that DNA-binding activities of cell envelopes and SDS-extracted envelopes represented that of the wild type cells responsible for flocculation. DNA-binding activities of cell envelopes were abolished by treating with proteases, suggesting that the DNAbinding factor is a proteinaceous component. Susceptibility of DNAbinding activities of SDS-extracted envelopes to 2,4,6-trinitrobenzene sulfonic acid or at high temperatures supported this assumption. Although cell envelopes prepared from the parent cells and mutant cells were subjected to SDS-polyacrylamide gel electrophoresis, no differences in protein composition were observed. Pseudomonas strain C-120, which was isolated from activated sludge, flocculates spontaneously after the late logarithmic growth phase, and floes are rapidly and completely deflocculated by treatment with added deoxyribonuclease (DNase).1) In a previous paper,2) we showed that the component responsible for flocculation was extracted from floes by treatment with 3m guanidine hydrochlo
The Journal of General and Applied Microbiology, 1987
The isoelectric points of serine/alkaline proteinases of various microorganisms reported to date ... more The isoelectric points of serine/alkaline proteinases of various microorganisms reported to date fall in the range of 4.9 to 10.7 (Table 1). Recent work in this laboratory (24) revealed that Aspergillus niger mutant 8, which is a non-acid-productive mutant, produces serine proteinases of an extremely anionic nature (p13.7). This stimulated the authors, who have been concerned with some ecological studies on microbial proteinases (25-27), to determining whether such novel serine proteinases of low p1 values might occur frequently in a group of fungi that, like the above mutant, produce no acid. Twenty-one strains of non-acid-productive fungi (Table 2) were used for the experiment. They were selected from the collection of this laboratory and from unidentified isolates using the surface colony technique of DAvis (28). About a half loopful of spores of each fungus was transferred from the slant to the surface of 1 Ecological Studies on the Proteolytic Enzymes of Microorganisms. Part IV.
Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and a... more Enterococcus faecalis is an opportunistic pathogen that causes serious infections in humans and animals and is also an important bacterium for dairy and probiotic supplement production. Therefore, bacteriophages infecting E. faecalis may be useful for phage therapy against multidrug-resistant strains or may threaten industrial fermentation. We isolated a virulent Siphoviridae bacteriophage, BC-611, specifically infecting E. faecalis strain NP-10011 but not infecting other E. faecalis strains or other enterococci. Although the genome sequence of BC-611 resembled that of enterococcal bacteriophage SAP6, BC-611 was marked by its narrow host specificity.
Promoter activity of the genes encoding Taka-amylase A and phosphoglycerate kinase of Aspergillus... more Promoter activity of the genes encoding Taka-amylase A and phosphoglycerate kinase of Aspergillus oryzae was analyzed using Escherichia coli ~glucuronidase as a reporter in a shoyu-koji mold A. oryzae KBN616. Assay of the ~ glucuronidase extracted from the mycelia of the transformants grown in the media containing different carbon sources suggests that expression of the Taka-amylase A gene was not induced by starch in A. oryzae KBN616. Analysis of proteins in the culture supernatant ofA. oryzae KBN616 after cultivation in starch medium supports this result. The phosphoglycerate kinase gene of A. oryzae KBN616 was shown to be expressed constitutively in the medium containing glucose and starch.
An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. Ther... more An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. There is a possibility that microorganisms are involved in generation of combustible gas from RDF and this study was aimed at showing the presence of bacteria that can ferment RDF pellets. All RDF samples tested contained a relatively high number of viable bacterial cells, 1:4 Â 10 5 to 3:2 Â 10 6 viable cells/g. These bacteria in the RDF samples fermented them to generate heat and hydrogen gas.
The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 ... more The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula  P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.
A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-... more A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-O-methyl-aD -glucuronosyl-xylotriose (MeGlcAX 3), as a main product from birchwood xylan and accumulated them in the culture under optimum conditions at pH 7.0 and 55°C using 0.75% (w/v) birchwood xylan as a carbon source for 42-72 h. The acidic xylotriose was purified by ethanol precipitation and high-performance liquid chromatography using NH 2 Lichosher® 100 column. The results of electrospray ionization mass spectrometry, mass to charge ratio (m/z) 603.23, confirmed that the purified sample was acidic xylotriose that had benefits and applications in many fields.
The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introd... more The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.
The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The struct... more The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.
The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open readin... more The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl -D-N,N-diacetylchitobioside [4-MU-(GlcNAc) 2 ]. The pH and temperature optima of the enzyme were 6.0 and 45°C, respectively. The K m and V max values for 4-MU-(GlcNAc) 2 were estimated to be 6.3 M and 46 mol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin. Chitinase (EC 3.2.1.14) is a glycosyl hydrolase that catalyzes the degradation of chitin, an insoluble linear -1,4-linked polymer of N-acetylglucosamine. Chitinases are present in a wide range of organisms, including bacteria, insects, viruses, plants, and animals, and play important physiological and ecological roles. On the basis of amino acid sequence homology, chitinases are divided into two unrelated families, families 18 and 19 of glycosyl hydrolases (15). Family 18 includes chitinases from bacteria, fungi, viruses, and animals and chitinases classified in class III or V from plants. On the other hand, family 19 includes almost exclusively plant chitinases classified in classes I, II, and IV but also a bacterial chitinase, Streptomyces griseus HUT 6037 chitinase C (32). To date, various chitinases were isolated from aerobic microorganisms such as Bacillus circulans (1, 55-57), Serratia marcescens (6, 13, 16, 20), an Aeromonas sp. (41, 51-53), an Alteromonas sp. (48), Streptomyces plicatus (37), Streptomyces olivaceoviridis (3, 36, 38), and Janthiobacterium lividum (12). Several genes encoding chitinases were cloned in Escherichia coli and characterized in detail along with their translated products (9, 12, 20, 36-38, 41, 43, 49, 56, 57). From these studies, chitinases were found to comprise two or more discrete domains, while the function of each domain has not yet been elucidated. Watanabe et al. reported that B. circulans ChiA1 has a chitin-binding domain (CBD) and two fibronectin type III (Fn3) domains in addition to a catalytic domain classified in family 18 (59). Deletion analysis indicated that a CBD but not Fn3 domains were responsible for binding of ChiA1 to insoluble chitins and further
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