• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.28-33
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• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.19-25
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• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• Journal of Animal Environmental Science
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• v.10 no.2
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• pp.111-118
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• 2004

This study analyzed temperature increase, microorganism changes, and inactivation of pathogenic microorganisms in pig slurry when treated with thermophilic microorganisms in Thermophilic Aerobic Oxidation(TAO) system. An amount of $6 m^3$ of pig slurry was treated in an $18 m^3(3.0\times2.5\times2.4 m)$ reactor for 5 to 7 days in two groups: the control of pig slurry only and the treatment of pig slurry with 6 liters of thermophilic microorganism(Bacillus sp.). To study the microorganism changes in the reactor, the populations of aerobic mesophilic microorganisms, thermophilic microorganisms and general pathogens were analyzed. To study the inactivation of pathogenic microorganisms, the levels of E. coli, Salmonella sp, Crytosporidium parvum and Giardia lamblia were analyzed. The temperature inside the reactor ranged from 18 to $62^{\circ}C$ for the control while far the treatment group it ranged from 18 to $66^{\circ}C$, showing a slightly higher array. With regard to changes in microorganisms, both mesophilic and thermophilic organisms decreased from $3.1\times10^6$ to $1.2\times10^2$ CFU/ml and from $1.0\times10^4$ to $8.0\times10^1$ CFU/ml, respectively, in the control. In the treatment, on the other hand, mesophilic organisms decreased from $3.0\times10^8$ CFU/ml to $8.6\times10^5$ CFU/ml while thermophilic organisms increased sharply from $2.0\times10^6$ to $1.2\times10^8$ CFU/ml. For pathogens, Salmonella and Giardia were not detected either before or after the treatment, while E. coli and C. parvum were found to be $10^5$ CFU/ml each before treatment and negative after it. From this experiment, it was concluded that thermophilic microorganisms could effectively sanitize liquid compost by generating high temperature in the TAO system, which in turn would inhibit the growth of pathogenic organisms.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.270-276
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• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Food Engineering Progress
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• v.14 no.1
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• pp.7-13
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• 2010

A thermophilic microorganism, which is able to hydrolyze starch, was isolated from soil and compost in Korea. It was Gram-positive, rod-shaped, catalase positive, nonmotile, glucose and mannitol fermentative, xylose oxidative, and spore forming microorganism. It also has an ability to hydrolyze casein and gelatin. The color of colony was yellowish white. The sequence of 16S rDNA of strain 2719 showed 99.5% sequence homology with the sequence of 16S rDNA of Bacillus thermoglucosidasius. On the basis of biochemical and physiological properties and phylogenetic analysis, the isolated strain was named as Bacillus thermoglucosidasius 2719.

• Korean Journal of Food Science and Technology
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• v.36 no.1
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• pp.105-110
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• 2004

Microorganism (strain DF 218) producing thermostable pretense was isolated from Korean soil and compost. It was Gram-positive, rod-shaped, aerobic, and spore-forming with yellowish white colony color, Temperature range for growth at pH 6.5 was $30-65^{\circ}C$, with optimum growth at $60^{\circ}C$. pH range for growth at $60^{\circ}C$ was 5-7 with optimum of 6.5, which indicates strain DF 218 to be thermophilic. The 16S rDNA sequence of strain DF 218 had 95% sequence similarity with that of Bacillus flexus. Based on physiological properties and phylogenetic analysis, we proposed the isolated strain as Bacillus sp. DF 218. Pretense was produced aerobically at $60^{\circ}C$ for 32 hr in a medium (pH 6.5) containing 1% each trypton, glucose, and NaCl. Its molecular weight was estimated as 61 kDa, with optimum temperature and pH of $60^{\circ}C$ and 7.5, respectively.

• Animal Bioscience
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• v.36 no.4
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• pp.671-678
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• 2023

Objective: Previous studies isolated the thermophilic ammonium-tolerant (TAT) bacterium Bacillus sp. TAT105 that grew in composting swine manure with the assimilation of ammonium nitrogen and reduced ammonia emissions during composting. Those studies also investigated the potential for applications of TAT105 to composting. It was observed that the concentration of TAT bacteria, phylogenetically close to TAT105, increased during composting. The objectives of this study were to identify the phylogenetic placement of these TAT bacteria and investigate their distribution in various composts. Methods: The phylogenetic placement of TAT105 was examined based on the sequence of 16S ribosomal RNA gene. The genomic DNA homology between TAT105 and the type strains of bacterial species that were phylogenetically close to TAT105 were examined by DNA-DNA hybridization. Moreover, the tolerances of these strains to NH₄Cl and NaCl were analyzed using a cultivation method. Concentrations of TAT bacteria in various composts were evaluated using an agar medium specific to TAT bacteria and polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results: TAT105 was most closely related to Bacillus thermolactis and Bacillus kokeshiiformis. Many variants of these species have been detected in various environments, including composts. The type strains of these species displayed TAT characteristics that were similar to those of TAT105. Among the composts examined in this study, TAT bacteria were detected at high concentrations (10⁵ to 10⁹ colony forming units per gram of dry matter) in most of the composts made from cattle manure, swine manure, bark, and excess sludge. Conclusion: TAT bacteria comprised B. thermolactis, B. kokeshiiformis, and their phylogenetically close relatives. They were considered to be adaptable to composting of some certain materials, and a favorable target for searching for strains with some useful function that could be applied to composting of these materials.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.662-671
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• 2015

To enrich the genetic resource of microbial xylanases with high activity and stability under alkaline conditions, a xylanase gene (xynSL4) was cloned from Planococcus sp. SL4, an alkaline xylanase-producing strain isolated from the sediment of soda lake Dabusu. Deduced XynSL4 consists of a putative signal peptide of 29 residues and a catalytic domain (30-380 residues) of glycosyl hydrolase family 10, and shares the highest identity of 77% with a hypothetical protein from Planomicrobium glaciei CHR43. Phylogenetic analysis indicated that deduced XynSL4 is closely related with thermophilic and alkaline xylanases from Geobacillus and Bacillus species. The gene xynSL4 was expressed heterologously in Escherichia coli and the recombinant enzyme showed some superior properties. Purified recombinant XynSL4 (rXynSL4) was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11. It had an apparent temperature optimum of 70℃ and retained stable at this temperature in the presence of substrate. rXynSL4 was highly halotolerant, retaining more than 55% activity with 0.25-3.0 M NaCl and was stable at the concentration of NaCl up to 4M. The enzyme activity was significantly enhanced by β-mercaptoethanol and Ca²⁺ but strongly inhibited by heavy-metal ions and SDS. This thermophilic and alkaline- and salt-tolerant enzyme has great potential for basic research and industrial applications.

• BMB Reports
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• v.32 no.5
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• pp.480-485
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• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• The Korean Journal of Mycology
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• v.45 no.1
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• pp.43-53
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• 2017

In this study, we analyzed the densities and taxonomic characteristics of various microorganisms that play important roles in Agaricus bisporus culture medium composting, and examined changes in the levels of decomposition-related enzymes secreted by these microorganisms. Various microorganisms such as thermophilic bacteria, actinomycetes, fluorescent Pseudomonas spp., and filamentous bacteria are closely associated with culture medium composts of Agaricus bisporus. The population densities of microorganisms change, and harmful bacteria disappear during thermophilic composting. Psychrobacter sp., Pseudomonas sp., Bacillus sp., and Pseudoxanthomonas sp. accounted for the highest proportion of bacteria in the culture media during outdoor composting, whereas Bacillus sp. and Psychrobacillus sp. were dominant after pasteurization. Cellulose and hemicellulose enzymes of the microorganisms were important at an early stage of rice straw composting and after decomposition of carbon sources, respectively. Microorganisms that secreted these enzymes were present in the second and third turning stage of composting.