• 생명과학회지
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• 제14권3호
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• pp.391-395
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• 2004

B. stearothermophilus NO2의 CGTase 유전자 (cgtS)를 구성적 $P_{JH}$ promoter 하류에 subcloning 하여 재조합 plasmid pIH-CGT1 (8.14 kb)을 구축하고 B. subtilis DB431에 형질 전환하였다. B. subtilis DB431/pJH-CGT1를 5가지 배지(LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG)로 flask 배양하여 균체증식과 CGTase발현량 및 분비국재성을 조사하여 최적 배지를 결정하였다. 그 중 〔5% molasses+2% CSL〕 배지에서 9시간에 1.8 unit/$m\ell$의 CGTase가 발현$.$생산되었다. 이 결과를 토대로 3. subtilis DB431/pJH-CGT1를 〔10% molasses + 5% corn steep liquor〕 배지에서 발효조 회분 배양한 결과, 30시간 배양시 CGTase의 최대 발현량은 4.2 unit/$m\ell$, 90%의 분비 효율, 90% 이상의 plasmid 안정성을 나타내었다. 저렴한 산업용 molasses 배지로 발효조 회분배양시 플라스크 배양보다 균체증식과 CGTase 발현량이 2배 이상의 증가된 값을 얻었다.

• 생명과학회지
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• 제14권1호
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• pp.121-125
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• 2004

E. coli에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현과 저온 배양의 공동작용 효과에 대해 조사하였다. 실험에 사용된 cgt와 groEL/ES 유전자를 발현하는 pTCGTl과 pGroll은 각각 T7 promoter와 Pzt-1 promoter에 의해 조절되고 이들을 E. coli에 도입시켰다. 대수증식기 초기(2 hr)와 대수증식기 중기(3 hr)에 tetrarycline 10 ng/ml 과 IPTG 1 mM을 첨가하여 각각의 유전자를 발현시켰다. CGTase활성과 specific artivity 측정 시 $37^{\circ}C$에서 pTCGTl 단독 발현 보다 $25^{\circ}C$에서 chaperone과 함께 발현시킨 경우 2배나 높은 활성이 측정됐으며, SDS-PACE 분석결과 $37^{\circ}C$에서 단독 발현 시킬경우 20% 정도 가용성 형태로 발현되던 것이 $25^{\circ}C$에서 chaperone과 공발현 시에는 거의 3.5배가 넘는 69%가 가용성으로 전환됨을 알 수 있었다. 이와 같이 분자 chaperone과 $25^{\circ}C$에서의 저온 배양은 E. coli에서 활성형질 가용성 CGTase의 생산 증가에 큰 영향을 미치는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.811-819
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• 1999

The ${\beta}-CGTase$ gene of alkalophilic Bacillus firmus var. alkalophilus was cloned into E. coli using $pZErO^{TM}-2$ as a vector. The cloned gene encoded a total of 710 amino acid residues consisting of 674 amino acids of the matured protein and 36 amino acids of the signal peptide, including 20 amino acids from the lacZ gene in the vector. Although the cloned ${\beta}-CGTase$ gene did not contain the promoter and start codons, it was expressed by the lac promoter and lacZ start codon in the $pZErO^{TM}$ vector. A comparison was made with the amino acid sequence and ten other CGTases from Bacillus sp. Also, ten highly conserved regions, which are important amino acid residues in catalysis of CGTase, were identified. The lac promoter used for expression of the ${\beta}-CGTase$ gene was induced constitutively in recombinant E. coli even without IPTG possibly because of a lack of the lacI gene in both host and vector, repressing the lacZ gene in the lac operon. Its expression was catabolically repressed by glucose, however, its repression was reduced by soluble starch, mainly because of the extremely high increase of the cAMP level. ${\beta}-CGTase$ can be overproduced in the recombinant E. coli by maintaining intracellular cAMP levels mostly through the intermittent feeding of glucose during cultivation.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.323-328
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• 1997

The recombinant Escherichia coli BL21(DE3)pLysE : pTCGT1 was grown to overproduce Bacillus macerans cyclodextrin glucanotransferase (CGTase) able to synthesize ${\alpha}$-cyclodextrin (CD) with a selectivity of 67%. A number of batch fermentations were performed to test the possibility of using lactose as an inducer of the E. coli T7 promoter system. A mixture of isopropyl ${\beta}$-D-thiogalactoside (IPTG) and lactose (1 : 1) gave a maximum CGTase activity of 2.4 U/ml, which was higher than the value obtained with induction by IPTG alone. Fed-batch fermentations involving a glucose-controlled growth period followed by a gene-expression phase with mixtures of IPTG and lactose were employed to achieve high cell density and thereby increase total CGTase activity. Optimized fed-batch fermentation using the modified inducer (IPTG : lactose=1 : 3) and 100 g/l yeast extract solution in the gene-expression phase resulted in a maximum CGTase activity of 62.9 U/ml and a final cell mass of 53.5 g/l, corresponding to a 31-fold increase in CGTase activity and a 29-fold increase in cell mass compared with the control batch fermentation.

• 한국미생물·생명공학회지
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• 제24권2호
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• pp.137-142
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• 1996

The biosynthesis and catabolite repression of cyclodextrin glucanotransferase(CGTase) and cyclodextrinase(CDase) were studied in Bacillus sp. KJI6. In accompanying to the cell growth, CGTase was synthesized during early growth phase (20h culture) and CDase was synthesized during late growth phase (60h culture). Synthesis of CGTase was rather constitutive than that of CDase in the absence or presence of carbon source. Production of CDase was strongly stimulated by amylopectin and $\gamma$-CD medium (about 6 times), but CGTase synthesis was slightly increased (about 1.3 times). Easily metabolizable carbohydrates such as D-glucose, D- fructose and D-mannose completely repressed the expression of CDase, whereas their repressive effect to CGTase synthesis was relatively negligible. By addition of 10 mM cAMP, any significant effect on the synthesis of the two enzymes was not observed. Hardly metabolizable glucose analogues such as 2-deoxy-D-glucose and 3-0-methyl-D-glucopyranose also did not show any repression on the syntheses of CGTase and CDase. This indicates that D-glucose has to be metabolized to exert its repressive effect. With these results, it seems likely that the biosynthesis of CGTase and CDase are regulated by the catabolite repression due to unknown metabolite(s) of EM pathway.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2003년도 International Meeting of the Microbiological Society of Korea
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• pp.122-124
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• 2003

Cyclodextrin glucanotransferase (CGTase) and endoxylanase genes of Bacillus sp. were subcloned down-stream of yeast ADH1 promoter and expressed in S. cerevisiae. Most of the CGTase and endoxylanase expressed were detected in the extracellular medium with activity of 0.6 and 7-8 unit/ml, respectively. The recombinant enzymes were secreted as N-linked-glycosylated forms, resulting an enhanced thermal stability. CGTase predominantly produced $\alpha-cyclodextrin$ from starch and endoxylanase produced xylobiose and xylotriose from xylan.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.206-209
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• 2002

Bacillus stearothermophilus의 CCTase 유전자(cgtS) 대장균과 효모의 shuttle vector로서 항구적 promoter인 adh l promoter를 함유한 pVT103-U(6.9Kb)에 도입하여 재조합 plasmid pVT-CCTS (9.0Kb)을 구축하고 효모 숙주 S. cerevisiae 2805에서 발현시켰다. 재조합 균주의 항구적 발현계인 2805/pv7-CGTS의 최적 발현조건은 YP배지에 dextrose 2%, pH 5.5, 30"C에서 최적 발효조건이었으며, CCTase의 최대 발현량은 48시간 배양시 0.624unit/mL을 나타내었다. B. stearothermophilus의 signal peptide가 재조합 효모에서도 높은 분비효율을 나타내어서 발현된 효소의 87%가 세포 외로 분비 생산되었다.산되었다.