• Korean Journal of Soil Science and Fertilizer
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• v.44 no.5
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• pp.836-840
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• 2011

In order to isolate thermophilic compost-promoting bacteria with high activity of cellulase and xylanase, spent mushroom substrates with sawdust were collected from mushroom cultivation farm, Jinju, Gyeongnam in Korea. Among of the isolates, one strain, designated SJ21 was selected by agar diffusion method. The strain SJ21 was identified as members of the Bacillus lincheniformis by biochemical characteristics using Bacillus ID kit and VITEK 2 system. Comparative 16S rDNA gene sequence analysis showed that strain SJ21 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus subtilis with 16S rDNA gene sequence similarity of 99%. On the basis of its physiological properties, biochemical characteristics and phylogenetic distinctiveness, strain SJ21 was classified within the genus Bacillus, for which the name Bacillus sp. SJ21 is proposed. The cellulase and xylanase activity of Bacillus sp. SJ21 was slightly increased according to bacterial population from exponential phase to stationary phase in growth curve for Bacillus sp. SJ21.

• The Journal of Natural Sciences
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• v.16 no.1
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• pp.15-29
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• 2005

To produce a thermostable cyclodextrin by using thermotolerant cyclomaltodextrin glucanotransferase(CGTase), a thermophilic and alkalophilic bacterium isolate, designated B-13 showing the highest CGTase activity was isalated from natural sources and identified as Bacillus cereus B-13 based on the morphological and physiological characteristics, and 16S rRNA sequence. The maximal CGTase activity (130 U/ml) was obtained when Bacillus cereus B-13 was cultured in SYC medium containing 2.0% soluble starch, 1.0% yeast extracts, 1% corn steep liquor and 1% $Na_2CO_3$ (pH 8.5) at $50^{\circ}C$ for 24 h and about 80% of maximal activity was also showed in he culture broth of $60^{\circ}C$ for 18 h. Optimum reaction temperature and pH of the partial purified CGTase for soluble starch were $65^{\circ}C$ and pH 8.5-9.0 respectively. The partial purified CGTase were also stable below $80^{\circ}C$ and pH 5.0-10.0. When 1% soluble starch was digested with the partial purified CGTase, the yield of cyclodextrin was 49%.

• 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.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1874-1883
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• 2008

Bacterial diversity and the composition of individual communities during the composting process of swine and mushroom cultural wastes in a field-scale composter (Hazaka system) were examined using a PCR-based approach. The composting process was divided into six stages based on recorded temperature changes. Phylogenetic analysis of eighty 16S rRNA sequences from uncultured composting bacterial groups revealed the presence of representatives from three divisions, including plant pathogenic bacteria, high-molecule-degrading bacteria and spore-forming bacteria. The plant pathogen A. tumefaciens gradually decreased in abundance during the composting process and eventually disappeared during the thermophilic and cooling stage. A bacterium homologous to Bacillus humi first appeared at the early thermophilic stage and was established at the intermediate thermophilic, post-thermophilic, and cooling stages. It was not possible to isolate the B. humi during any of the stages using general culture techniques.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.5
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• pp.516-521
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• 2014

A bacterium producing ${\alpha}$-galactosidase (${\alpha}$-$\small{D}$-galactoside galactohydrolase, EC 3.2.1.22) was isolated. The isolate, KM-1 was identified as Bacillus coagulans based on its 16S rRNA sequence, morphology, and biochemical properties. ${\alpha}$-Galactosidase activity was detected the culture supernatant of B. coagulans KM-1. The bacterium showed the maximum activity for hydrolyzing para-nitrophenyl-${\alpha}$-$\small{D}$-galactopyranoside (pNP-${\alpha}Gal$) at pH 6.0 and $50^{\circ}C$. It hydrolyzed oligomeric substrates such as melibiose, raffinose, and stachyose liberating a galactose residue, indicating that the B. coagulans KM-1 ${\alpha}$-galactosidase hydrolyzed ${\alpha}$-1,6 linkage. The results suggest that the decreased stachyose and raffinose contents in fermented soybean meal are due to the ${\alpha}$-galactosidase activity.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• Journal of Microbiology and Biotechnology
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• v.22 no.5
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• pp.592-599
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• 2012

Bacillus licheniformis ${\alpha}$-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.

• Korean Journal of Soil Science and Fertilizer
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• v.48 no.5
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• pp.485-491
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• 2015

This study was performed to investigate thermophilic bacteria from soil having broad antifungal spectrum against Rhizoctonia solani, Colletotrichum gloeosporioides, Phytophthora capsici, Fusarium oxysporum f.sp. lycopersici, and Botrytis cinerea. One isolate selected could resist heat shock of $60^{\circ}C$ for one hour, and had broad antifungal activity in dual culture assay against all tested fungal pathogens and was identified as Bacillus amyloliquefaciens Y1 using 16S rRNA gene sequence. Further investigation for antifungal activity of bacterial culture filtrate (BCF) and butanol crude extract (BCE) of various concentrations showed broad spectrum antifungal activity and fungal growth inhibition significantly increased with increasing concentration with highest growth inhibition of 100% against R. solani with 50% BCF and 11 mm of zone of inhibition against R. solani with 4 mg BCE concentration. Treatment of butanol crude extract resulted in deformation, lysis or degradation of C. gloeosporioides and P. capsici hyphae. Furthermore, B. amyloliquefaciens Y1 produced volatile compounds inhibiting growth of R. solani (70%), C. gloeosporioides (65%) and P. capsici (65-70%) when tested in volatile assay. The results from the study suggest that B. amyloliquefaciens Y1 could be a biocontrol candidate to control fungal diseases in crops.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.7
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• pp.1045-1054
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• 2012

The phylogenetic diversity of the bacteria in hot composting samples collected from three spatial locations was investigated by molecular tools in order to determine the influence of gradient effect on bacterial communities during the thermophilic phase of composting swine manure with rice straw. Total microbial DNA was extracted and bacterial near full-length 16S rRNA genes were subsequently amplified, cloned, restriction fragment length polymorphism-screened and sequenced. The superstratum sample had the highest microbial diversity among the three samples which was possibly related to the surrounding conditions of the sample resulting from the location. The results showed that the sequences related to Bacillus sp. were most common in the composts. In superstratum sample, 45 clones (33%) and 36 clones (27%) were affiliated with the Bacillus sp. and Clostridium sp., respectively; 74 clones (58%) were affiliated with the Clostridium sp. in the middle-level sample; 52 clones (40%) and 29 clones (23%) were affiliated with the Clostridium sp. and Bacillus sp. in substrate sample, respectively. It indicated that the microbial diversity and community in the samples were different for each sampling site, and different locations of the same pile often contained distinct and different microbial communities.

• 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.