• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.411-416
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• 2002

Bacillus macerans cyclodextrin glucanotransferase (CGTase) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein, Aga1p. The surface display of CGTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form ${\alpha}$-cyclodextrin from starch. The maximum surface-display of CGTase was obtained by growing recombinant S. cerevisiae at $20^{\circ}C$ and pH 6.0. S. cerevisiae cells displaying CGTase on their surface consumed glucose and maltose, inhibitory byproducts of the CGTase reaction, to enhance the purity of produced cyclodextrins. Accordingly, the experimental results described herein suggest a possibility of using the recombinant S.cerevisiae anchored with bacterial CGTase on the cell surface as a whole-cell biocatalyst for the production of cyclodextrin.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.442-449
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• 1998

CGTase를 이용한 당전이 xylitol의 합성과 당전이 xylitol의 비피더스균 생육증식 효과에 대한 연구를 수행하였다. 수종의 세균류가 분비하는 CGTase의 xylitol에 대한 당전이능을 비교하였으며, Thermoanaerobacter sp. 유래의 CGTase가 가장 우수한 당전이능을 보였다. 각종 당공여체를 검토한 결과 압출전분이 가장 우수한 결과를 보였으며, 당전이 효소반응의 최적 조건을 검토하였다. 생성된 당전이 xylitol을 활성탄-셀라이트 칼럼 크로마토그래피를 이용하여 분리하여 두 개의 fraction인 F-I, F-II를 얻었다. 이들의 당쇄결합 양상을 FAB mass spectrometer와 $^{13}$C-NMR spectrometer, 그리고 glucoamylase을 이용한 효소소화법을 이용하여 분석한 결과 xylitol에 glucose와 maltose 분자가 $\alpha$-1,4 결합되어 있는 것으로 유추되었다. 얻어진 당전이 xylitol은 xylitol과는 달리 Bifidobacterium breve에 대한 생육촉진효과를 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.656-659
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• 2001

Cyclodextrin glucanotransferase (CGTase) (EC 2.4.1.19) use starch to produce cyclic maltooligosaccharides (cyclodextrins, CDs) which are of interest in various applications. To obtain a novel CGTase having high CD-forming activity, ${\beta}-cyclodextrin$ glucanotransferase $({\beta}-CGTase)$ from Bacillus firmus var. alkalophilus was modified through site-directed mutagenesis and constructed five mutants, H59T, H59Q, Y96M, 9O-PPI-93, and ${\Delta}(148-154)D$, respectively. Y96M and ${\Delta}(148-154)D$ showed much higher level of conversion yields of starch into CDs from 28.6% to about 39% compared to wild-type ${\beta}-CGTase$, respectively, but 90-PPI-93 maintained similar convesion yields of starch to CDs. And their ${\beta}-CD$ ratios to total CDs were not changed and maintained, and convesion yields to linear maltooligosaccharides of all mutants were not changed significantly. These results indicates that five mutations of ${\beta}-CGTase$ from Bacillus firmus var. alkalophilus appears to be important roles for increase of overall CD production rather than change of its product specificity, especially.

• 한국미생물·생명공학회지
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• 제21권5호
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• pp.461-467
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• 1993

Transglycosylation reaction of cyclodextrin glucanotransferase (CGTase) was analyzed in the attrition coupled heterogeneous reaction system using raw starch as a donor` and mono-, di-saccharide, and glycoside as acceptors. For transglycosylation reaction of stevioside, the transglycosylation rate was similar and the transglycosylation yield was increased compare with conventional process using liquefied starch as the donor. Also the accumulation of maltooligosaccharides in reaction mixture was minimized.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.671-674
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• 2001

정제, 농축된 free CGTase 는 pH 6.0 - 7.0의 범위에서 안정하였으며, $70^{\circ}C$정도의 고온에서도 안정한 특성을 가지고 있었으며, CGTase의 고정화를 위한 방법으로서 CNBr-activated sepharose 4B에 의한 고정화가 가장 높은 효율올 나타내는 것으로 결정되었다. 고정화의 최적조건은 $30^{\circ}C$, 60rpm, pH 6.0의 phosphate buffer 하에서 9 시간 동안 고정화하는 것이었고, CNBr-activated sepharose 4B를 이용한 고정화의 경우 실험에 사용된 다른 모든 이온교환 수지에서 얻어진 효율에 비해 월등한 효과를 보였다.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.274-281
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• 1996

A bacterium producing Cyclodextrin Glucanotransferase (CGTase) and Cyclodextrinase (CDase) was isolated from soil, and named as Bacillus stearothermophilus KJ16. The growth of the isolated strain occurred in two steps, and syntheses of CGTase and CDase were dependedt on the growth cycle of the cell. CGTase was constitutively synthesized during the 1st growing phase, while CDase was synthesized inducibly during the 2nd growing phase. When the midium pH was controlled at 7.0 the maximum enzyme activities of CGTase and CDase were increased by 12-fold (1300 mU/ml) and 2-fold (225 mU/ml), respectively, compared with the pH-uncontrolled batch culture. The CGTase of the isolate converted soluble starch to CDs with the ratio of $\alpha$-CD:$\beta$-CD:$\gamma$-CD=42:46:12 at $55^{\circ}C$.The optimal pH and temperature of CGTase were 6.0 and $60^{\circ}C$, respectively and the optimal pH and temperature of CDase were 6.0 and $55^{\circ}C$. The molecular weights of the purified CGTase and CDase were estimated to be 65, 000 and 68, 000 dalton, respectively. Among several substrates, $\gamma$-CD was most rapidly hydrolyzed by the purified CDase.

• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.62-69
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• 1999

In order to investigate the critical amino acid residues involved in the catalytic activities of $\beta$-cyclodextrin glucanotransferase ($\beta$-CGTase) excreted by Bacillus firmus var. alkalophilus, the amino acid residues in $\beta$-CGTase were modified by various site-specific amino acid modifying reagents. The cyclizing and amylolytic activities of $\beta$-CGTase were all seriously reduced after treatment with Woodward's reagent K (WRK) modifying aspartic/glutamic acid, N-bromosuccinimde (NBS) modifying tryptophan, and diethylpyrocarbonate (DEPC) modifying histidine residues. The roles of tryptophan and histidine residues in $\beta$-CGTase were further investigated by measuring the protection effect of various substrates during chemical modification, comparing protein mobility in native and affinity polyacrylamide gel electrophoresis containing soluble starch, and comparing the $K_m$ and $V_{max}$ values of native and modified enzymes. Tryptophan residues were identified as affecting substrate-binding ability rather than influencing catalytic activities. On the other hand, histidine residues influenced catalytic ability rather than substrate-binding ability, plus histidine modification had an effect on shifting the optimum pH and pH stability.

• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.191-193
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• 2000

B. macerans 유래의 CGTase를 yeast surface display기술을 이용하여 S. cerevisiae의 표면에 발현된 것을 halo-test와 immunofluorescence microscopy와 flow cytometry를 통하여 확인하였다. 재조합 효모는 효소의 cyclization작용을 저해하고 CD의 분해작용을 촉진하는 glucose와 maltose를 제거하는 발효공정과 표면 발현된 CGTase의 cyclization 공정을 동시에 수행할 수 있어 CD의 생산, 분리공정을 효율적으로 개선하였다.