• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.34-45
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• 2012

A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.272-279
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• 2006

The gene encoding Thermus sp. T351 alkaline phosphatase (T351 APase) was cloned and sequenced. The gene consisted of 1,503 bp coding for a protein with 500 amino acid residues including a signal peptide. The deduced amino acid sequence of T351 APase showed relatively low similarity to other Thermus APases. The T351 APase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21 (DE3). The expressed enzyme was purified by heat treatment, and $UNO^{TM}$ Q and $HiTrap^{TM}$ Heparin HP column chromatographies. The purified enzyme exhibited high activity at extremely alkaline pHs, reaching a maximum at pH 12.0. The optimum temperature of the enzyme was $80^{\circ}C$, and the half-life at $85^{\circ}C$ was approximately 103 min. The enzyme activity was found to be dependent on metal ions: the addition of $Mg^{2+}$ and $CO^{2+}$ increased the activity, whereas EDTA inhibited it. With p-nitrophenyl phosphate as the substrate, T351 APase had a Michaelis constant ($K_{m}$) of $3.9{\times}10^{-5}M$. The enzyme catalyzed the hydrolysis of a wide variety of phosphorylated compounds.

• Korean Journal of Environmental Biology
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• v.25 no.3
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• pp.260-266
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• 2007

A thermophilic bacterium capable of poly (L-lactide)(PLLA) degradation was isolated from cultivating soil in Korea. The isolate was Gram positive rod-shaped bacterium, and was identified as Geobacillus caldoxylosilyticus based on the 16S rDNA sequence analysis. The strain proved to be a new PLLA degrading bacterium which has not been reported in the open literatures yet. The degradation activity of the strain was assessed in a sterilized compost inoculated with the strain under controlled compost condition at $58^{\circ}C$ for 40 days. The strain mineralized 66%, 57%, 41% and 40% of PLLA5000, PLLA11000, PLLA34000 and PLLA256000 whose weight average molecular weights were 5000, 11000, 34000 and 256000, respectively. Incorporation of 0.1% each of gelatin, yeast extract and ammonium sulfate in the compost containing PLLA256000 as a nutritional supplement raised the biodegradation activity by 27%, 13% and 10%, respectively. Increase of the inoculum size from $10^9cfu\;g^{-1}\;to\;10^{10}cfu\;g^{-1}\;and\;10^{11}cfu\;g^{-1}$ also enhanced the biodegradation activity by 14% and 20%, respectively.

• Journal of Life Science
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• v.25 no.12
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• pp.1450-1457
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• 2015

To overcome the depletion of fossil fuels and environmental problems in future, the research and production of biofuels have attracted attention largely. Thermophilic microorganisms produce effective and robust enzymes which can hydrolyze plant biomass and survive under harsh bioprocessing conditions. Caldicellulosiruptor bescii, which can degrade unpretreated plants and grow on them, is the one of the best candidates for consolidated bioprocessing (CBP). C. bescii can hydrolyze pectin efficiently as well as the major plant cell wall components, cellulose and hemicelluloses. Many glycosyl hydrolases and carbohydrate lyases with multidomain structure play an important role in plant biomass decomposition. Recently genetic tools for metabolic engineering of C. bescii have developed and bioethanol production from unpretreated biomass is achieved in C. bescii. Here, we review the recent studies for biomass degradation by C. bescii and bioethanol production in C. bescii in order to provide information about metabolic engineering of themophilic bacteria and biofuel development.

• Journal of Life Science
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• v.12 no.2
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• pp.129-135
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• 2002

Several kinds of thermophilic, aerobic microorganisms (Bacillus genus), metal leaching microorganisms (Thiobacillus, T. ferooxidans), and other nondegradable chemical-degrading microorganisms (Pseudomonas genus) were utilized to study the effect on composting livestock manure. Under the Carbon-Nitrogen ratio (C/N) of 35∼40 and water content of 50∼65% conditions, the composting in the cycling drum reactor showed slower composting and lower temperature increase than that of the manual reactor. Element analysis after composting indicated relatively high levels of mineral contents with the substitutional effect of chemical fertilizer. Metal analysis before and after composting showed lower As in all, Cr in pig, Pb in cow, Hg in chicken, and Cu in cow manure compost than the regulation values. Compost maturity was ascertained by the several maturity tests. Salmonella and E. cozi detection test by SS or EMB agar plate confirmed the safety from the pathogenic microorganisms. The results suggest that the inoculation of metal and some other chemical degrading microorganisms during composting might decrease metal contamination and increase composting rate.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.271-279
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• 1992

Genomic DNA of Bacillus stearothemzophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindIII, cloned into pBR322, and subsequently transferred into the Escherichra coli HB101 cells. Three among 5, 000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids (pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindIII fragment originated from B. stearothemzophilus which was responsible for the xylanase activity. pMGl3, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.

• Journal of Conservation Science
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• v.7 no.1
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• pp.31-41
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• 1998

Two isolation methods using the open plate and air sampler were applied to examine microbial distribution as an environmental factor for conservation of cultural properties in the War Memorial in Korea. The numbers of fungi were the abundant in the air of entrance and inside of the exhibition room where visitors were crowded, compared with inside of repository rooms. Fungal and bacterial distributions in the exhibition room during exhibition period were higher than during nonexhibition period, These results are due to the inflow of air current carrying microoganisms to the exibition rooms from outside with visitors. Fungi isolated from the War Memorial were identified as 19 species and one thermophilic fungus, Neurospora crassa. The majority of the isolated fungi are seemed to cause damage to cellulose materials in the Memorial.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.136-141
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• 1988

Susceptibility of a thermophilic strain (D-71) of Saccharomyces cerevisiae and an osmotolerant strain (SR-S) of Zygosaccharomyces rouxii to Zymolyase-20T were studied in various renditions. Content of glucan and mannan in cell wall of Saocharomyces cerevisiae D-71 were 14.5% and 14.8%, and Zygosaccharomyces rouxii SR-S were 24.0% and 19.0%, respectively. Susceptibility of Saccharomyces cerevisiae D-71 cultured in Wickerham synthetic medium containing 0.5% of methionine and 0.1% of glucose to Zymolyase-20T was 66%, and $K_2$HPO$_4$ and aminobenzoic acid were greatly effective to susceptibility. Susceptibility of Zygosaccharomyces rouxii SR-S cultured in Wickerhnin synthetic medium containing 0.5% of peptone, 0.15% of methionine and 0.l% of glucose to Zymolyase-20T was 80%, and KI and pyridoxine were greatly effective to susceptibility. Susceptibility of Saccharomyces cerevisiae D-71 stationary cultured in YMPG medium at $25^{\circ}C$ for 12 hours was 16o1e and Zygosaccharomyces rouxii SR-S stationary cultured in YMPG medium at $25^{\circ}C$ for 30 hours was 82%.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.925-934
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• 2010

Three typical biological solid wastes, namely, animal manure, garbage, and sewage sludge, were compared with regard to the composting process and the changes in microbial community structure. The effects of different bulking agents such as rice straw, vermiculite, sawdust, and waste paper were compared in manure compost. The differences in the microbial community were characterized by the quinone profile method. The highest mass reduction was found in garbage composting (56.8%), compared with manure and sludge (25% and 20.2%, respectively). A quinone content of $305.2\;{\mu}mol/kg$ was observed in the late stage of garbage composting, although the diversity index of the quinone profile was 9.7, lower than that in manure composting. The predominant quinone species was found to be MK-7, which corresponds to Gram-positive bacteria with a low G+C content, such as Bacillus. The predominance of MK-7 was especially found in the garbage and sludge composting process, and the increase in quinones with partially saturated long side-chains was shown in the late composting process of manure, which corresponded to the proliferation of Actinobacteria. The effects of different bulking agents on the composting process was much smaller than the effects of different raw materials. High organic matter content in the raw materials resulted in a higher microbial biomass and activity, which was connected to the high mass reduction rate.