• 생명과학회지
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• 제8권3호
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• 미생물학회지
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• 제36권3호
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• pp.203-208
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• 2000

재조합 플라스미드 pAL850에 함유된 Bacillus circulans KCTC3004 $\alpha$-amylase 유 전자를 pUB110을 이용하여 shuttle 플라스미드 pALS111을 만들어 Bacillus 세포에 이동. 발현시켰다. Bacillus subtilis(고초균)와 Bacillus megaterium(거대균)으로 형질전환된 pALS111로부터 $\alpha$-amylase는 세포증식과 비례하여 생산되었다. 형질전화주가 생산하는 $\alpha$ -amylase의 최대활성을 유전자 공여 균주인 B. circulans와 비교했을 때 고초균은 약 95배, 거대균은 약 34배 정도의 높은 활성을 나타내었다. 그리고 대장균 형질전환주는 분비율이 10% 정도인데 반하여 고초균 형질전화주는 생산된 효소전부를 , 거대균 형질전환주는 약 98%를 세포외로 분비함을 보임으로써 고초균과 거대균은 실용적인 면에서 대장균보다 우월 함을 나타내었다, pALS111의 각 숙주 내에서의 안정성을 살펴본 결과 거대균에서는 92%, 고초균에서는 76%, 대장균에서는 38% 로 나타났다. SDS-PAGE와 zymogram을 통해 추정 된 대장균과 고초균, 거대균에서 발현된 효소의 분자량은 약 55,000으로 확인되었다. 이들 형질전환주가 생산하는 $\alpha$-amylase는 starch 에 작용하여 주된산물로서 maltotriose 이상의 다양한 maltooligosaccharide들을 생산함이 확인 되었다.

• Preventive Nutrition and Food Science
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• 제7권3호
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• pp.310-316
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• 2002

Extracellular cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. JK-12 was purified through sev-eral purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE-sephadex A-50 and Mono QIM HR5/5. The purified CGTase exhibited a single band on SDS-PAGE and was estimated to be approximately 82 kDa. The isoelectric point of the enzyme was 7.2 as determined by isoelectric focusing. The CGTase from Paenibacillus sp. JK-12 had a transglucosylation activity at the C-2 position of L-ascorbic acid. The optimum pH and temperature for the CGTase activity were 8.0 and 5$0^{\circ}C$, respectively. The enzyme activity was stable from pH 6.0 to 9.() and at temperatures up to 55$^{\circ}C$ at pB 8.0, having 80% residual activity. The activity of the CGTase was strongly resistant to metals such as A $g^{+}$ and $Ba^{2+}$ but slightly inhibited by H $g^{+}$, N $i^{2+}$ and $Mg^{2+}$. The enzymeproduced $\alpha$ -cyclodextrin ($\alpha$-CD) and $\beta$-CD as the main products from starch, but not ${\gamma}$-CD.X>-CD.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.61-67
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• 1995

To investigate the role of induction on CGTase production for alkalophilic Bacillus firm us var. alkalophilus H609, the constitutive mutants that form a halo around its colonies at non-inducible AG agar media containing amylose and glucose were selected. The selected constitutive mutants could produce CGTase in the range of 18.9 to 28.8 units/ml $\cdot A_{600}$ in the alkaline basal medium, and finally a constitutive mutant Bacillus firmus var. alkalophilus CM46 was selected. The constitutive nature of CM46 was also confirmed in protein level using SDS-PAGE. The effects of induction and catabolite repression for both parent strain Bacillus firmus var. alkalophilus H609 and constitutive mutant CM46 were also compared by adding soluble starch and glucose during cultivation. The selected mutant CM46 was a non-inducible but a catabolite regulated type mutant. Even though inductive regulation was released, the specific CGTase activity defined as CGTase activity per cell concentration was not increased compared with that of parent strain. The cell growth and CGTase production patterns of constitutive mutant Bacillus firmus var. alkalophilus CM46 were compared with the parent strain to identify CGTase production characteristics.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.242-250
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• 2001

A Gram-positive bacterium (strain JB-13) that was isolated from soil as a producer of cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] was identified as Panibacillus sp. JB-13. This CGTase could catalyze the transglucosylation reaction from soluble starch to L-ascorbic acid (AA). A main product formed by this enzyme with ${\alpha}-glucosidase$ was identified as $2-O-{\alpha}-D-glucopyranosyl{\;}_{L}-ascorbic$ acid (AA-2G) by the HPLC profile and the elemental analysis. CGTase was purified to homogeneity using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Seohadex A-50, and gel chromatography on Sephacryl S-200HR. The molecular weight was determined to be 66,000 by both gel chromatography and SDS-PAGE. The isoelectric point of the purified enzyme was 5.3. The optimum pH and temperature was PH 7.0 and $45^{\circ}C$ respectively. The enzyme was stable in the range of pH 6-9 and at temperatures of $75{\circ}C$ or less in the presence of 15 mM ${CaCl_2}.\;{Hg^2+},\;{Mn^+2},{Ag^+},\;and\;{Cu^2+}$ all strongly inhibited the enzyme's activity.

• 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.

• 생명과학회지
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• 제8권4호
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• pp.387-394
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• 1998

A thermophilic bacterium (strain DG0303) producing a thermostable $\alpha$-glucosidase was isolated from manure and identified as Bacillus sp. Strain DG0303 produced high level of $\alpha$-glucosidase compared with other thermophilic Bacillus strains. The cellular protein patterns were also compared with other Bacillus strains by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE). On the basis of 16S rDNA analysis the Bacillus sp. DG0303 was found to be a member of Bacillus rDNA group 5. The optimum temperature for growth was 65$\circ$C and no growth was obtained at 40$\circ$C or 75$\circ$C. The optimum pH for growth was 5.5 to 8.5. $\alpha$-glucosidase activity was produced during growth and most activity was detected in the culture supernatant. The $\alpha$-glucosidase production was constitutive in the absence of carbohydrates. High level of enzyme activity was detected when the culture was grown on medium containing starch. Addition of glucose resulted in the repression of the $\alpha$-glucosidase production. The optimum pH and tempoerature for enzyme activity were pH 5.0 and 65$\circ$C, respectively. When analyzed by zymogram, the culture supernatant showed a single $\alpha$-glucosidase band with a molecular weight of approximately 60,000.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1555-1563
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• 2008

A novel $\alpha$-amylase ($\alpha$-1,4-$\alpha$-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal a-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and $55^{\circ}C$ with an apparent $K_m$ value of 1.66 mg/ml and $V_{max}$ of 0.1${\mu}mol$glucose $min^{-1}$ $ml^{-1}$. ScAmy43 activity was strongly inhibited by $Cu^{2+}$, $Mn^{2+}$, and $Ba^{2+}$, moderately by $Fe^{2+}$, and was only weakly affected by $Ca^{2+}$ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and $\beta$-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.

• 한국식품과학회지
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• 제30권6호
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• pp.1426-1431
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• 1998

본 연구에서는 재조합 Bacillus subtilis에서 발현된 Streptomyces albus KSM-35유래의 amylase를 정제하고 특성을 구명하였다. 정제된 효소는 SDS-PAGE를 통하여 분자량이 약 50 kD인 것으로 밝혀졌으며, isoelectric focusing을 통하여 측정된 pI값은 약 4.3이었다. 효소의 최적 반응온도는 $45^{\circ}C$이었으며 최적의 pH는 6.0이었다. D-value는 45, $55^{\circ}C$에서 각각 279분, 191분이었고 D-value로부터 계산된 Z-value는 $17.7^{\circ}C$였다. 수용성 전분용액을 기질로 사용한 효소반응의 초기에는 maltotriose, maltopentaose와 maltotetraose가 주로 생성되었지만 시간이 경과함에 따라 이들의 농도는 감소하였고 maltose의 농도가 점차 증가하였다. 이러한 반응 생성물의 분해는 Thin layer chromatography를 통하여 확인할 수 있었다. 기질에 의한 저해가 없다고 가정하고 Michaelis-Menten kinetics를 이용하여 속도상수를 추정하였을 때 최대 반응속도는 0.37 mM/min, Michaelis-Menten 상수는 0.13% (w/v)로 나타났다.

• 한국미생물·생명공학회지
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• 제26권4호
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• pp.323-330
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• 1998

호알카리성 Bacillus firmus var. alkalophilus가 생산하는 cyclodextrin glucanotransferase(CGTase)를 호화시킨 팽윤감자전분을 이용한 흡착 및 탈착, 한외여과, DEAE-cellulose 이온교환 크로마토그래피, 그리고 Sephacryl HR-100을 이용한 gel filtration 등의 분리정제 과정을 통하여 정제하였다. 정제된 CGTase의 분자량은 약 77,000 Da이었으며, 등전점은 6.2～6.3이었다. 최적 효소반응 온도 및 pH는 각각 5$0^{\circ}C$ 와 6.0 였고, 열 및 pH 안정성은 5$0^{\circ}C$까지와 pH 6.0~9.5 사이였다, 정제된 CGTase는 $Ca^{2+}$ 에 의하여 열 안정성이 크게 상당히 향상되었으며 최적 효소반응 온도와 열 안정성도 55~6$0^{\circ}C$와 6$0^{\circ}C$로 증가하였다. CGTase의 기질 특이성을 검토한 결과 여러 종류의 전분들로부터 $\beta$-CD를 주로 생성하였으며, ${\gamma}$-CD도 소량 생성하였으나 $\alpha$-CD는 거의 생성되지 않았다. Sweet potato starch, corn starch, 그리고 amylopectin을 기질로 할 때 높은 CD전환율을 보였으며 $\beta$-CD와 ${\gamma}$-CB의 생성비가 5.8~8.4:1로서 $\beta$-CD를 고 수율로 생산하는 전형적인 $\beta$-type의 CGTase였다. 또한 본 효소의 최적 CD 생산조건은 기질농도 5.0%(w/v) corn starch, 효소첨가량 25 unit/g of starch로서 반응 8시간 후의 전체 CD량도 약 25 g/l로서 전환율은 50%였고, 이 때 $\beta$-CD농도는 21 g/l로서 전체 CD 중 84% 였다.