• 자연과학논문집
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• 제16권1호
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• pp.15-29
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• 2005

내열성의 cyclomaltodextrin glucanotransferase (CGTase)를 이용하여 열에 안정한 싸이클린 텍스트린 (CD)을 생산하기 위하여 매우 높은 CGTase 활성을 보이는 고열성이며 호알카리성인 B-13세균을 분리하여 형태적, 생리학적 특성과 16S 리보솜 RNA 서열분석 등을 통하여 Bacillus cereus B-13으로 동정하였다. Bacillus cereus B-13 2% 가용성 전분, 1% 효모 추출물, 1% $Na_2CO_3$ 등을 함유하는 SYC 배지 (pH 8.5)에 접종하여 $50^{\circ}C$에서 24시간 배양하였을 때 최고의 CGTase 활성(130 U/ml)을 보였다. 또한 부분 정제된 CGTase의 작용 최적 온도와 pH는 각각 $60^{\circ}C$, pH 8.5-9.0 이었고 $80^{\circ}C$이하와 pH5.0-10.0에서 안정하였다. 1% 가용성 전분을 부분 정제한 CGTase와 작용시켰을 때 49%의 CD 수율을 보였다.

• Journal of Microbiology and Biotechnology
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• 제3권2호
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• pp.78-85
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• 1993

In order to elucidate the mechanism which regulates the production of cyclodextrin glucanotransferase (CGTase) and to achieve overproduction of CGTase by releasing catabolite (glucose) repression, several catabolite repression resistant mutants were selected from newly screened Bacillus firmus var. alkalophilus H609, after NTG (N-methyl-N -nitro-N-nitrosoguanidine) treatment, using 2-deoxyglucose as a nonmetabolizable analog of catabolite glucose and as a selection marker. Five catabolite repression resistant mutants were selected from about 30, 000 2-deoxyglucose resistant colonies. Relative catabolite repression indices of the selected mutants were in the range of 8~80% assuming 100% for parent strain. The amount of CGTase produced by the mutant strain CR41, which was 250 units/ml, was three times larger than that produced by its parent strain. The mutation seems to have occurred in the regulatory region of CGTase gene and not in the structural region or the glucose transporting system in cell membrane. The enzymatic properties of CGTase excreted from parent and mutant strains were also compared.

• 한국미생물·생명공학회지
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• 제23권1호
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• pp.31-35
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• 1995

Using the alkalophillic Bacillus sp. SH-8 and its mutant Bacillus sp. SH-8M capable of growing at the neutral pH, the amino acid compositions of the cell wall and cell membrane were studied at varying cultivation pH's. The pattem of protein electrophoresis was also tested. It was elucidated that the amino acids consisting of the cell wall were alanine, glutamic acid, lysine, aspartic acid, and meso-diaminopimelic acid. There was not any significant difference in the amino acid compositqon betweeo`two straqns regardless of the culture pH. As the results of HPLC ssay, glutamic acid and aspartic aciu accounted for more than 50% in the amqno acid composytqon of the cell wall. By the isolatqon of the crude cell membrane and the SDS-PAGE analysis, it was found that there was a considerable difference qn the protein pattern when the straqns were cultured at the neutral pH. In addition, by the two dimensional gel electrophoresis, it was confirmed that there was a difference in the protein patterns between two strains cultivated at the neutral pH medium but no difference at the alkaline medium.

• Journal of Applied Biological Chemistry
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• 제49권4호
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• pp.135-139
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• 2006

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.206-212
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• 1995

A strain BL-29, which produces a extracellular lytic enzyme on E. coli was isolated from the soil. The strain was identified as belonging to the genus Bacillus sp. The lytic enzyme was purified to homogeneity by ion exchange chromatography and gel filtration. Specific activity of the purified enzyme was 28, 850 U/mg protein and yield of the enzyme was 5$%$. The purified enzyme showed a single band on SDS-PAGE and its molecular weight was estimated to be 31, 000 by SDS-polyacrylamide gel electrophoresis and gel filtration column chromatography. The optimum temperature and pH were $55^{\circ}C$ and pH 10.0, respectively. The enzyme was stable at $45^{\circ}C$ but enzyme activity was reduced by up to 50$%$ when the temperature was raised to $55^{\circ}C$ for 15 min. Stable range of pH was from 5.0 to 11.0. but Enzyme activity was inhibited by lead-acetate, mercuric chloride, ethylene glycol-bis-[$\beta$-aminoethyl ether]-N, N, $N^1, $N^1$-tetraacetic acid (EGTA), and ethylenediamine tetraacetic acid (EDTA), but not affected considerably by treatment with other chemical reagents.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.759-765
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• 2004

The NCS inhibited the activity of glucosyltransferase which was produced by Streptococcus mutans JC-2, and the rate of inhibition at $100\muM<$ and $200\muM$ were 74.0% and 99.8%, respectively. It was stable in alkali condition, but unstable in acid condition. It was also stable up to $80^{\circ}C$. The kinetic study of the inhibition by NCS was carried out by Lineweaver-Burk plot and Dixon plot. It was non-competitive inhibition, determined by the two plots and $K_i$ and $K_i$ values were $15\muM$ and $19.3\muM$ respectively. The NCS did not show cytotoxicity against human gingival cells at $K_i$ ($15\muM$, $150\muM$, $1,500\mu$ M) concentrations. It had less cytotoxicity than chlohexidin, which has usually been used as the agent of anticaries. To evaluate the industrial applicability of the NCS, human pluck tooth was used. The inhibitory rates of tooth calcification and calcium ion elution by the NCS were 41 % and 2.5 times, respectively. These results suggested that NCS from Bacillus sp. S-1013 is an efficient anticaries agent.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.753-759
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• 2002

$\beta$-Cyclodextrin glucanotransferase ($\beta$-CGTase) was overproduced extracellularly using recombinant E. coli by transforming the plasmid pECGT harboring a secretive signal peptide. The $\beta$-CGTase gene of alkalophilic Bacillus firmus var alkalophilus was inserted into the high expression vector pET20b(+) containing a secretive pelB signal peptide, and then transformed into E. coli BL2l(DE3)pLysS. The optimum culture conditions fer the overproduction of $\beta$-CGTase were determined to be TB medium containing 0.5% (w/v) soluble starch at post-induction temperature of $25^{\circ}C$. A significant amount of $\beta$-CGTase, up to 5.83 U/ml, which was nine times higher than that in the parent strain B. firmus var. alkalophilus, was overproduced in the extracellular compartment. A pH-stat fed-batch cultivation of the recombinant E. coli was also performed to achieve the secretive overproduction of $\beta$-CGTase at a high cell density, resulting in production of up to 21.6 U/ml of $\beta$-CGTase.

• 한국미생물·생명공학회지
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• 제22권1호
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• pp.59-64
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• 1994

The production and the characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were investigated under the different pH conditions. Bacillus sp. SH-8 and Bacillus sp. SH-8M showed the maximum activity of protease at 60 hours(70 units/ml) AND 96 houre(50 units/ml) cultivation, respectively, under the alkaline condition(pH 10.2). However, Bacillus sp. SH-8M exhibited the maximum activities in 8 days cultivation at pH 6.9 and in 6 days cultivation at pH 7.7 Bacillus sp. SH-8M showed the protease activity at the pH change from alkaline to neutral condition, whereas Bacillus sp. SH-8 did not. In addition. all the enzymatic characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were similar with the regardless of different pH conditions.

• 한국식품과학회지
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• 제40권1호
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• pp.70-75
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• 2008

두 개의 amylase를 호알칼리성 Pseudomonas sp. KFCC 10818의 배양액으로부터 정제하여 그 특성을 조사하였다. 정제된 효소의 분자량은 각각 50 kDa과 75 kDa이었다. 이 효소들의 최적반응온도는 각각 $35^{\circ}C$와 $40^{\circ}C$였으며 50 kDa의 효소는 칼슘이온에 의하여 효소활성이 두 배로 촉진되었다. 이 두 효소는 최적 pH가 6-8 부근이었으며 pH 12의 조건에서도 효소활성을 유지하는 알칼리내성을 나타냈다. Maltooligosaccharide이나 soluble starch로부터 maltose와 maltotriose를 최종 효소반응산물로 생산하였다. 두 amylase는 N-말단 아미노산 서열이 각각 QTVPKTTFV와 DTVPGNAFQ로 분석되었다.

• KSBB Journal
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• 제8권3호
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• pp.209-216
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• 1993

미생물을 이용한 생분해성 고분자 생산연구의 일환으로 토양으로부터 고점성의 생물고분자 생산균주를 분리하고, 이 분리균에 대한 분류학적 성질 및 생성 생물고분자의 이화학적 성상을 검토, 규명하였다. 분리균주는 Alcaligenes속의 한 균종으로 동정하였으며, 호알카리성 균주의 특성을 나타내었다. CPC(cetylpridinium chloride)침전 및 acetone처리하여 정제한 산성 고분자 물질은 carboxyl기를 갖고 자외 흡수가 강한 glutamic acid만을 구성성분으로 하였고, glutamic acid는 $\gamma$-peptide 결합의 $\gamma$-polyglutamic acid로 존재하였다. 중화당량은 약 350으로 $\gamma$-PGA 2.7잔가당 1개의 음이온이 존재하였으며, 분자량은 약 $6.5{\times}10^5$Daltons이었다.