• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.244-250
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• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Proceedings of the Korean Society of Life Science Conference
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• 한국생명과학회 2002년도 제35회 학술심포지움 및 춘계학술대회
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• pp.83-83
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• 2002

• Journal of Applied Biological Chemistry
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• 제50권4호
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• pp.196-201
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• 2007

An intracellular invertase was purified to homogeneity from the cell extract of an alkalophilic and thermophilic Bacillus sp. TA-11, which was classified as a new species belonging to Bacillus cereus based on chemotaxanomic and phylogenetic analyses. The purified enzyme with a recovery of 26.6% was determined to be a monomeric protein with a molecular weight of 23 kDa by SDS-PAGE and 26 kDa by gel filtration. The maximum enzyme activity was observed at pH 7.0 and $50^{\circ}C$, and the purified enzyme was stable at the pH range of 5.0 to 8.0 and below $60^{\circ}C$. $K_m$ and $V_{max}$ values of the enzyme for sucrose were 370 mM and 3.0 ${\mu}M$ per min, respectively. The enzyme activity was significantly inhibited by bivalent metal ions ($Hg^{2+}$, $Cd^{2+}$ and $Cu^{2+}$) and sugars (glucose and fructose).

• Korean Journal of Soil Science and Fertilizer
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• 제44권5호
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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.

• Korean Journal of Food Science and Technology
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• 제23권1호
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• pp.31-36
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• 1991

Cellulase genes from thermophilic alkalophilic Bacillus sp. F204 a potent cellulase complex-producing bacterium, were cloned in Escherichia coli with pUC 19. Plasmids pBC191 and pBC192, isolated from transformants forming yellow zone around colony on the LB agar plate containing 0.5% carboxymethyl cellulose and ampicillin, contained 4.6 Kb and 5.8 Kb HindIII fragments, respectively. The 4.6 Kb insert of pBC191 had single sites for BamHI EcoRI, KpnI and pvuII. DNA hybridization and immunodiffusion studies showed that pBC191-encoded cellulase gene was homologous with that of host strain. pKC231, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pKK223-3, E. coli expression vector, and pGC711, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector, had 3.2 times and 2.8 times as much cellulase activity as pBC191, respectively. Substrate specificity analysis showed that cellulases cloned were CMCase.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.153-156
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• 2002

The characterization of a partially purified, cellulase-free, thermostable alkaline xylanase from thermoalkalophilic Bacillus sp. JB-99 was investigated. The xylanase production was the highest when birchwood xylan was added to a medium containing finely powdered rice bran, showing 4,826 IU$ml^-1$ of activity for 15 h of incubation. The partially purified xylanase exhibited an optimum temperature and pH at $70^C{\circ}$ and 10, respectively. The enzyme was stable at pH 5-11 at $50^C{\circ}$. The xylanase activity was strongly inhibited by $Hg^2+$, while dithiothreitol, cysteine, and ${\beta}$-mercaptoethanol enhanced the activity.

• Microbiology and Biotechnology Letters
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• 제17권5호
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• pp.436-439
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• 1989

The chromosomal DNA fragments of thermophilic alkalophilic Bacillus sp, K-17, a potent xylanhydrolyzing bacterium, were ligated to a vector plasmid pBR322 and transformed into Escherichia coli HB101. The plasmid pAX278, isolated from a transformant forming yellow color on the LB agar plate containing 1 mM p-nitrophenyl- $\beta$-xylopyranoside, was found to enable the transformants to produce p-xylosidase. The 5.0 kilobase insert of pAX278 had single sites for EcoRI, PstI, XbaI, and PvuII, and 2 sites for BglII. Biotinylated pAX218 was hybridized to 0.9 kb as well as 5.0 kb fragment from Bacillus sp. K-17 DNA on nitrocellulose filter. pGX718 was constructed by inserting the 5.0 kb HindIII fragment of pGX278 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector. The enzymatic properties of $\beta$-xylosidase from E. coli HB101 carrying recombinant plasmid were the same those of $\beta$-xylosidase from Bacillus sp. K-17.

• Microbiology and Biotechnology Letters
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• 제34권4호
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• pp.363-367
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• 2006

Food wastes were decomposed into the Mugri (Isung Engineering, Korea), a food waste reduction machine, with adding sawdust of cryptomeria. Degradation effects were better when the machine worked at over 45$^{\circ}C$ than those at the lower temperature. Thermophilic bacteria were isolated from cryptomeria sawdust and the food waste products degraded by the machine. The isolates from cryptomeria sawdust were classified into 3 genera (Acinetobacter baumannii, Enterobacter sp. and Erwinia cypripedii) and almost all the isolates from the degraded products were partially identified as Bacillus sp. by 16S rDNA sequence analysis. The isolated thermophilic bacteria showed degradative enzyme activities. In the case of addition of the 30 thermophilic bacteria into the machine, degradation rate of food wastes was almost twice as high with increasing process temperature up to 6$^{\circ}C$.

• Asian-Australasian Journal of Animal Sciences
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• 제25권7호
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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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• 제8권1호
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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.