• Applied Biological Chemistry
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• v.40 no.6
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• pp.465-471
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• 1997

The cyclodextrin glycosyltransferase (CGTase, EC 3.2.1.19) from Bacillus brevis CD162 was purified by precipitating with ammonium sulfate, DEAE-Sepharose CL-6B column chromatography and Sephadex G-150 column chromatography. The molecular mass and pI of the purified enzyme were estimated to be 74,000 and 6.3 by SDS-PAGE and isoelectric focusing, respectively. The purified enzyme was clearly identified as the CGTase by zymogram after SDS-PAGE. The optimum pH and temperature for the enzyme activity were 8.0 and $55^{\circ}C$, respectively. The enzyme was stable at the range of pH $5.5{\sim}9.0$, and up to $50^{\circ}C$. The amino acid sequence from the $NH_2-terminal$ of the purified CGTase was Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln. The yields of the products from starch as the substrate were 1.3% for ${\alpha}-$, 33.9% for ${\beta}-$, and 9.7% for ${\gamma}-cyclodextrin$.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.459-464
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• 1997

A cyclodextrin glycosyltransferase-producing bacterium was newly isolated from soil using alkaline pH medium containing 1% $Na_2CO_3$. The isolated strain was identified as Bacillus brevis by morphological and biochemical characteristics, and fatty acid composition and designated Bacillus brevis CD162. The strain showed the best enzyme production of 0.9 unit/ml after 96 hrs of culture at $30^{\circ}C$ in a medium of 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% $K_2HPO_4$ 0.05% $MgSO_4{\cdot}7H_2O$, and 1.5% $Na_2CO_3$ at initial pH 10.2.

• Korean Journal of Food Science and Technology
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• v.25 no.6
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• pp.608-613
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• 1993

Production of cyclodextrin (CD) by cyclodextrin glycosyltransferase (CGTase) isolated from alkalophilic Bacillus sp. was carried out to determine optimal reaction conditions. The maximum initial rate of CD production from amylose was obtained at dextrose equivalent 10.5. The CD production yield showed inverse proportionality to DE values over the range from 0.5 to 37.7. Even though the deactivation constant of CGTase at $60^{\circ}C$ was higher than those at lower temperatures, the production rate and yield at $60^{\circ}C$ were still higher. These results suggest thermal stabilization of CGTase by binding with starch.

• Korean Journal of Food Science and Technology
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• v.23 no.4
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• pp.503-509
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• 1991

Action of a cyclodextrin glycosyltransferase (CGTase) produced from alkalophilic Bacillus sp. No.4 was studied in a solution containing starch and sucrose to prepare glycosyl sucrose syrup with good sweetness and antidecaying properties of teeth. In the initial stage of the reaction the CGTase produced cyclodextrin, however, the cyclodextrin disappeared and glycosyl sucrose was formed with the lapse of reaction time. The best proportion of sucrose to starch for prodution of glycosyl sucrose was about 1 : 1. The optimum pH and temperature of the coupling reaction was pH 6.0 and $60^{\circ}C$, respectively. Main composition of glycosyl sucrose syrup prepared with 20% starch and 20% sucrose was sucrose 18%, glucosyl sucrose ($G_{2}F$) 15.3% and maltosyl sucorse ($G_{3}F$) 11.3%. And glucose, maltose and maltotriose were produced very little. Smaller amounts of acid and insoluble glucan were formed in the syrup by Streptococcus mtans OMZ176 than in the sucrose. Therefore, the prepared glycosyl sucrose sucrose syrup is expected to prevent teeth from decaying.

• Korean Journal of Food Science and Technology
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• v.26 no.4
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• pp.375-381
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• 1994

A bacterial strain No. KY-126, which produced extracellular cyclodextrin glycosyltransferase(CGTase), was isolated from soil and identified as Bacillus stearothermophilus KY-126. The enzyme was purified by the treatments of ammonium sulfate precipitation, DEAF-Sephadex, Sephadex G-100 column chromatography. The optimal pH and temperature for the enzyme activity were pH 5.5 and $65^{\circ}C$, respectively. And the enzyme was stable at pH values from 6.0 to 11.0 at $55^{\circ}C$ for 30 min and stable up to $60^{\circ}C$ for 30 min.. The enzyme was inhibited by $HgCl_{2}$. The molecular weight of the enzyme was estimated to be 67,000 by using SDS-PAGE. The maximum conversion from starch to cyclodextrin (CD) by CGTase was 43% and obtained at 6 hr reaction and the ratio of ${\alpha}-,\;{\beta}-,\;{\gamma}-$, CD production at this time was 2.9 : 2.1 : 1.0.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.44-48
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• 1990

Alkalophilic sp. YC-335 isolated from soil was capable of producing large amount of cyclodextrin glycosyltransferase (CGTase) in culture broth. This enzyme was successively purified 52.9 folds with 17.8 yield by ethanol precipitation, DEAE-Toyopearl column chromatography and Sephadex G-100 column chromatography. The purified enzyme have a molecular weight of approximately 75,000 estimated by SDS polyaerylamide gel electrophoresis. The optimum pH and temperature for the enzyme activity were 6.0 and 5$0^{\circ}C$, respectively. The enzyme stable between pH 6 and 10, and up to 5$0^{\circ}C$. The thermostability of the enzyme was increased up to 6$0^{\circ}C$ by the addition of 15mM CaCl$_2$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.2
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• pp.351-357
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• 1997

The cyclodextrin glycosyltransferase(EC 3.2.1.19) from Bacillus firmus was purified by precipitating with ammonium sulfate followed by, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 column chromatography. In this way, we were able to obtain the single band protein on SDS-PAGE with a yield of 12%, whose purity was 49 fold. The purified CGTase was identified as a protein having molecular weight of approximately 80,000 dalton and isoelectric point of 9.6. The optimum pH and temperature for the enzyme activity were 8.0 and $65^{\circ}C$, respectively. The enzyme was stable at between pH 5.5 and 9.0 and up to $50^{\circ}C$. After 24hr of enzyme reaction using soluble starch as substrate, the ratio of ${\alpha}-$, ${\beta}-$ and ${\gamma}-cyclodextrin$ production was 0.01 : 2.90 : 1.00, respectively. And this CGTase pro-duced mainly ${\beta}-$ and ${\gamma}-cyclodextrin$.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.725-729
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• 2008

An enzyme reactor installed with ultrafiltration membrane was developed to produce ${\alpha}-,\;{\beta}-$, and ${\gamma}$-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTKIOase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a ${\alpha}$-CD yield of 24.0 (${\pm}1.5$)% and a productivity of 4.68 (${\pm}0.88$) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1-nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.92-96
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• 2001

Effect of environmental factors on the expression of soluble forms of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21(DE3)pLysE:pTCGT1 were investigated. The amount of soluble CGTase produced in the cell was measured by determining its enzymatic activity. The soluble fractionof the enzyme was increased by lowering the culture temperature to $30{\circ}C$ and medium pH to 5.8 compared to the enzyme production in LB medium at $37^{\circ}C$ and pH7.0. Addition of 0.2 M NaCl enhanced enzyme expression levels at the expense of cell growth. Glycine betaine that was added after 3 h of induction protected not only the cell growth from hig osmotic pressue but also hepld in vivo folding of CGTase in recombinant E. coli. Addition of 1 mM $CaCl_2$ was also effective in the expression of soluble CGTase, resulting in 15 U/ml of the enzyme activity.

• BMB Reports
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• v.40 no.3
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• pp.333-340
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• 2007

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower $K_m$ for coupling reaction using cellobiose and cyclodextrins as substrates.