• 생명과학회지
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• 제11권2호
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• pp.190-195
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• 2001

The cuclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned at the downstream of yeast ADH1 promoter, and then the resulting plasmid pVT-CGTM(9.15 kb) was introduced into the yeast host strain, Saccharomyces cerevisias 2805. The transformed yeast, S. cerevisiae 2805/pVT-CGTM, showed the starch-hydrolyzing activity on the starch-azure plate. The optimal conditions for the CGTase expression were found to be 2% dextrose, initial pH5.5, 3$0^{\circ}C$, and 48hr cultivation. Under this condition, the extracellular CGTase activity reached at 0.53 U/mL, whereas the intracellular activity was about 0.03U/mL. This result indicates that the signal peptide of Bacillus CGTase functioned well in S. cerevisiae.

• 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.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.29-36
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• 2004

토양으로부터 CGTase를 생산하는 새로운 호열성 세균을 분리하였으며, 형태 및 생리적 특성과 16S ribosomal DNA의 염기서열을 분석한 결과 Geobacillus thermosacchalytycus 로 동정하였다. 호열성 G. thermosacchalytycus가 분비하는 CGTase를 한외여과, 소수성 Phenyl Sephadex CL-4B 클로마토그래피 , 그리고 gel filtration의 순서로 분리 정제하여 정제도 28.2배, 정제수율 12.2%, 그리고 specific activity가 4952.5 units/mg인 CGTase를 얻을 수 있었다. 정제된 CGTase의 분자량은 69,000 dalton이었고, terminal amino sequence는 Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr-Ser-Asp-Val-Val-Tyr-Gln-Ile이었다. 최적 pH 및 온도는 각각 pH 6와$ 50^{\circ}C$였고, pH 6-8과 $60^{\circ}C$에서 장기간 보존할 수 있는 중성 pH의 내열성 효소임을 알 수 있었다. 내열성 CCTase의 cyclization과 coupling 반응의 특정을 검토한 결과 G. thermosacchalytycus가 생산하는 내열성 효소는 $\alpha$-type CGTase이었고, cyclization 반응보다는 coupling반응에 적합한 당전이성이 높은 효소임을 알 수 있었다. 당전이 반응에서 기질 특이성을 검토한 결과 당공여체로는 가용성 전분 그리고 당수용체로는 배당체인 stevioside에 대해 높은 기질 특이성을 보였다. 당전이 반응은 당수용체와 당공여체가 효소와 무작위로 결합하여 진행되는 random ternary complex mechanism으로 반응이 수행되었다.

• 한국식품영양과학회지
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• 제43권9호
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• pp.1388-1393
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• 2014

선행연구에서 얻은 alkalophilic Bacillus I-5 유래의 CGTase wild-type과 가수분해능이 강화된 mutant 효소를 활용하여 waxy rice starch로부터 아밀로펙틴 클러스터를 제조하였다. SEC-MALLS-RI 분석법으로 CGTase wild-type과 mutant 효소가 처리된 시료의 평균 분자량을 확인한 결과 10분가량의 효소반응으로 두 반응 모두 평균 분자량은 $10^4{\sim}10^5Da$으로 급격히 감소하였음을 확인하였으며, 일정 반응 시간이 경과한 이후에는 더 이상 분자량의 감소가 일어나지 않음으로 미루어 시료가 아밀로펙틴 클러스터 단위로 분해되었으며 그 분자량은 $10^4{\sim}10^5Da$ 정도임을 알 수 있다. 또한 MALDI-TOF/MS 분석을 통하여 CGTase wild-type은 다양한 종류의 cyclic 형태의 maltodextrin을 생성하고 있으며 mutant 효소는 주로 소량의 maltooligosaccharide 들을 생산함을 확인하였다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1550-1553
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• 2007

The functional characteristics of a ${\beta}$-cyclodextrin glucanotransferase (CGTase) excreted from alkalophilic Bacillus sp. BL-31 that is highly specific for the intermolecular transglycosylation of bioflavonoids were investigated. The new ${\beta}$-CGTase showed high specificities for glycosyl acceptor bioflavonoids, including naringin, rutin, and hesperidin, and especially naringin. The transglycosylation of naringin into glycosyl naringin was then carried out under the conditions of 80 units of CGTase per gram of maltodextrin, 5 g/l of naringin, 25 g/l of maltodextrin, and 1 mM $Mn^{2+}$ ion at $40^{\circ}C$ for 6 h, resulting in a high conversion yield of 92.1%.

• Applied Biological Chemistry
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• 제47권1호
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• pp.41-48
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• 2004

Cyclodextrin glucanotransferase(CGTase)를 생성하는 Bacillus sp. 균주를 토양으로부터 분리하였으며, CGTase 생성을 위하여 0.1% albumin, 2% $NH_4Cl$, 2% soluble starch, 0.2% $NH_2PO_4$를 효소생산배지에 첨가하여 $37^{\circ}C$에서 72시간 배양 시 최대의 활성을 나타내었다. Sephadex G-100과 G-150을 사용한 gel filtration과 DEAE-cellulose를 이용한 이온 교환 크로마토그래피로 9.72배 정제하였으며, specific activity는 528.02 unit/mg이었다. CGTase의 효소학적 특성은 최적 pH, 최적 온도는 pH 8.0과 $80^{\circ}C$였으며, pH $8.0{\sim}11.0$과 $60{\sim}80^{\circ}C$에서 안정하였다. 금속이온 중 $Pb^{2+},\;Hg^{2+}$와 $Zn^{2+}$에서 효소활성이 억제되었다. CGTase의 첨가 효소량을 $20.5{\sim}410$ unit 까지 변화시키며 stevioside로의 당전이능을 조사한 결과 20.5 unit 및 41 unit 처리 시 당전이율은 74.9%와 75,7%로 높게 나타났으며, 205 unit 처리시에는 당전이율은 68.7%로 비슷하였으나 갈변화가 진행되었다. 효소의 농도를 높여 410 unit를 처리했을 때 당전이율은 57.9%로 더욱 떨어졌으며 역시 갈변화가 진행되어 제품의 물성을 나쁘게 하였고, 당전이 산물 중 pentaglycoside와 hexaglycoside가 검출되지 않아 필요이상의 효소사용량은 오히려 당전이 반응에 역효과가 나타났다.

• 생명과학회지
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• 제12권6호
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• pp.688-693
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• 2002

Chaperone 분자는 세포 내에서 새로 합성된 polypeptides의 misfolding을 보호하는 역할을 가진다. 이런 chaperone 분자와의 공발현은 활성형 재조합 단백질의 생산을 증가를 기대할 수 있다. 본 연구에서는 E. cozi에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현의 효과에 대해 조사하였다. cgt와 groEL/ES 유전자출 발현하는 pTCGT1과 pGro7은 각각 T7 promoter와 araB promoter에 의해 조절되고 이들을 E. coli cell에 co-transformation시켰다. 재조합 E. coli에서 IPTG와 L-arabinose의 최적 농도를 결정하기 위해 행한 결과 1 mM IPTG, 0.3 mg L-arabinose/$m\ell$에서 가장 높은 CGTase 활성을 나타내었다. 그리고 tube에서는 L-arabinose와 IPTG를 각각 0.4~0.5 $OD_{600}$과 0.8~l.0 $OD_{600}$에서 첨가하였을 때 활성형 CGTase의 생산이 증가되었다. GroEL/ES 공발현 조건에서는 가용성 CGTase 활성이 0.7~0.73 unit/$m\ell$로 단독 발현의 0.36~0.56 unit/$m\ell$에 비해 약 1.5 배 정도 증가함을 알 수 있었다. SDS-PAGE 분석에서는 GroEL/ES 공발현 조건에서 총 CGTase의 33.6%정도가 가용성 형태로 생산됨을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제8권2호
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• pp.152-157
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• 1998

To analyze the role of the C and D domains in the cyclization activity of cyclodextrin glucanotransferase (CGTase), two plasmids, pKB1ΔC300 and pKB1ΔD96, were constructed in which DNA regions encoding 100 and 32 amino acids, respectively, from the C and D domains of B. stearothermophilus NO2 CGTase were deleted. The mutated CGTase from the pKBlΔC300 produced much lower amounts of ${\alpha}$-, ${\beta}$-, and $\gamma$-cyclodextrin (CD) than the parental CGTase. However, the mutated CGTase from the pKBlΔD96 showed a similar production pattern of CDs to wild-type CGTase. The production ratios of the ${\alpha}$-, ${\beta}$- and $\gamma$-CDs were not affected by the deletions, when compared to those of parental CGTase. The optimum temperature of the mutated CGTase from the pKBlΔC300 was decreased from $60^{\circ}C$ to $55^{\circ}C$. The optimum pH of the mutated CGTase from the pKB1D96 was shifted from 6.0 to 7.0. The thermostability of the two mutant CGTases were not changed. From these results, it is suggested that the C and D domains are not related to cyclization activity directly because mutant-enzymes deleted C or D domains still possessed their activity. However, they are important for other enzymatic properties such as productivity and pH optimum as a partition of CGTase tertiary structure.

• 한국미생물·생명공학회지
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• 제19권2호
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• pp.171-178
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• 1991

The two-stage enzyme reactor, packed with cyclodextrin glucanotransferase (CGTase) immobilized on Amberite IRA 900, coupled with ultrafiltration membrane was investigated for continuous production of cyclodextrin (CD). 5% (w/v) of soluble starch was partially cyclized, in the 0.1 l first-stage immobilized enzyme reactor, up to CD conversion yield of 10% (w/w) at retention time of 0.56hr and 1.5 units of immobilized CGTase/1g of carrier. In the second stage main immobilized enzyme reactor capacity of 1.5 l, the maximum CD conversion yield of 39% (w/v) was achieved at retention time of 2.8hr and 0.47 unit of CGTase/1 g of carrier. Unreacted residual dextrin was fractionated with ultrafiltration membrane, and then, recycled into the second-stage main bioreactor to increase the CD conversion yield. The most suitable membrane size and the volume concentration ratio (concentrate: filterate) for recycling of unreacted residual dextrin were found to be 5K dalton and 4:6, respectively. CD conversion yield was increased about 3~4% upon co-immobilization of pulluanase along with CGTase. Spent Amberite IRA 900 can be reutilized consecutively more than 3 times for immobilization of CGTase after regeneration.