• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.54-62
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• 1991

Cyclodextrin glucanotransferase(CGTase) derived from Bacillus macerans was immobilized by (1) covalent linkage on chitosan and chitin with glutaraldehyde, (2) adsorption on DEAE-cellulose and Amberite IRA 900 after succinylation, and (3) entrapment on alginate and polyacrylamide by cross linking. Adsorption on Amberite IRA 900 and covalent linking on chitosan were identified to be the most suitable immobilization methods considering the yield of activity and stability of immobilized CGTase. The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble CGTase. Thermal stability of CGTase immobilized on chitosan was increased from 50 to $55^{\circ}C$, and the optimum temperature of CGTase immobilized on Amberite IRA 900 was shifted from 55 to $50^{\circ}C$. The effect of molecular size of soluble starch (substrate) on immobilized CGTase investigated using partially liquefied substrates with different dextrose equivalent(DE). Cyclodextrin(CD) conversion yield augmented according to the increase of DE level for immobilized CGTase on Amberite IRA 900. CD conversion yield of partially cyclized starch with soluble CGTase was higher compared with liquefied one with ${\alpha}-amylase$.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.1002-1005
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• 2002

The effects of GroEL/ES chaperone on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli were investigated. The cgt gene and groEL/ES genes are under the control of T7 promoter and Pzt-1 promoter, respectively. The optimal concentrations of inducers, IPTG and tetracycline, were found to be 1.0 mM and 10 ng/ml, respectively. When tetracycline and IPTG were added at the early exponential phase (2h) and exponential phase (3h) of growth, respectively, about 1.5-fold increase of soluble CGTase activity and 1.6-fold increase of soluble CGTase protein were obtained. An SDS-PAGE analysis revealed that about $37.2\%$ of total CGTase protein was in the soluble fraction when GroEL/ES chaperone was overexpressed.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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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%.

• Microbiology and Biotechnology Letters
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• v.25 no.3
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• pp.305-310
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• 1997

For the continuous production of transglucosylated steviosides, cyclodextrin glucanotransferase from Bacillus macerans was immobilized onto Diaion HPA 75 (styrene-divinylbenzene resin) that was screened from ion exchange resins, synthetic adsorbents and chitosan derivatives. The parameters influencing enzyme immobilization were examined in order to maximize the activity of immobilized enzyme. The optimum conditions for immobilization turned out to be: contact time 2 hr at 30$circ$C, pH 6$sim$9, and enzyme loading 20mg protein/g resin at 4.4 Os/Kg as osmolarity. Competing with other molecules having low molecular weight, enzyme was immobilized reversibly. The activity of immobilized enzyme was as high as 180U/g resin when the diafiltrated solution of stock enzyme was used. The optimum conditions for transglucosylation were as follows: pH 6.0, temperature 50$circ$C, 30% substrate solution composed of 15% stevioside mixture and 15% dextrin of which value of dextrose equivalent was about 9.0.

• Korean Journal of Food Science and Technology
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• v.26 no.1
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• pp.6-11
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• 1994

A cyclodextrin glucanotransferase(CGTase)-producing Bacillus sp. I-5 was isolated from soil and the enzyme exhibited the maximum reaction rate at pH 8.0 and $50^{\circ}C$. It was found that CGTase of I-5 produced ${\beta}-$ and ${\gamma}-CD$ mainly but the production ratio of cyclodextrins (CDs) was influenced by the buffer solution. Sodium acetate significantly stimulated the formation of ${\gamma}-CD$, increasing the content by 35%. The production of CDs was influenced by DE value of starch. The results indicated that DE value in the range of $3.5{\sim}6.0$ were most effective for the CD formation. CGTase was immobilized on the reversibly soluble-insoluble carrier, hydroxypropyl mothylcellulose acetate succinate. The immobilized CGTase was soluble at pH 7.5, and precipitated easily at pH 6.0. Enzyme reactor was designed to produce CD continuously. It was composed of three major stages-CD produttion by immobilized CGTase, conversion of the residual dextrin to glucose by amylase and glucoamylase and alcohol fermentation by yeasts to remove the glucose into alcohol. The yield of total CDs was 3.65g from 10g soluble starch.

• Journal of Life Science
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• v.11 no.2
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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 Life Science
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• v.14 no.5
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• pp.794-799
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• 2004

Cyclodextrin glucanotransferase (CGTase) produced by Bacillus subtilis NAl/pKBl was used for the production of cyclodextrin (CD). The enzyme was purified by ion exchange and gel filtration chromatography. The purified enzyme exhibited its maximum activity in the pH range of 6.0 to 7.0 and temperature range of 60 to $70^{\circ}C$. Immobilization of purified CGTase was carried out with various immobilization matrices. Amberlite IRA-900, a strong basic anion exchange resin, showed the highest immobilization ability (38 units per gram resin). Optimal pH and temperature for enzymatic reaction of the immobilized CGTase were pH 6.0 and 60t. The activity of immobilized CGTase maintained more than a month and could be reused for a month in a continuous enzyme reactor for the production of CD.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.461-466
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• 1986

A highly cyclodextrin glucanotransferase producing strain of Bacillus sp. was isolated from soil, and basic studies on the characteristics of the strain and its enzyme, conditions for the enzyme production, and the enzyme utilization were carried out. The isolated strain was aerobic, motile, endospore-forming and rod-shaped bacterium. Optimum pH and temperature for the enzyme action were 6.0 and 45$^{\circ}C$, and the enzyme was stable within 5$0^{\circ}C$, and between pH 6.0 and 10.0. The highest yield of the enzyme was obtained using the medium containing 2% corn starch as a carbon source, and 5% corn steep liquor, 0.1% urea and 0.25% ammonium sulfate as nitrogen sources. The fermentation conditions for the enzyme production in a jar fermentor were cetermined to be 3$0^{\circ}C$, 200rpm, 0.6vvm and 60hr cultural period. Stevioside transglycosylation catalyzed by this enzyme was identified by high performance liquid chromatography.

• Preventive Nutrition and Food Science
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• v.7 no.3
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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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• v.9 no.2
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• pp.230-233
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• 1999

A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.