• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.156-163
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• 1992

Bacillus sp. was isolated from soil for its strong activity of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19). The enzyme was purified by gel filtration and anion exchange column chromatography using FPLC. The purified enzyme exhibited its maximum CGTase activity in the pH range of 6~8 and the temperature range of 50~$70^{\circ}C$. The molecular weight was estimated as 114,000 by SDS-PAGE. The isoelectric point of the enzyme was 4.3. The CGTase of Bacillus sp. E l produced $\beta$-cyclodextrin mainly and did not produce a-cyclodextrin. The product ratio of $\beta$-cyclodextrin to $\gamma$-cyclodextrin was 7:l.

• Microbiology and Biotechnology Letters
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• v.21 no.2
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• pp.119-124
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• 1993

An alkalophilic microorganism for overproduction of cyclodextrin glucanotransferase (CGTase) was newly isolated from hot-water spring soil, and identified as Bacillus firmus var. alkalophilus H609. The strain maintained stability during preservation and cultivation for the enzyme production, and produced significant amount of CGTase corresponding to the volumetric activity of 75 units/mL at 37C, initial pH of 11.2, and after 40 hours. The strain excreted several different proteins showing CGTase activity that catalyzed the formation of mainly beta-and Gamma-type cyclodextrin (ratio of 7:1) from soluble starch without accumulation of alpha-type. Other enzymatic properties were also investigated.

• Journal of Life Science
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• v.8 no.3
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.293-297
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• 2002

The cyclodextrin glucanotransferase (CGTase) gene from Bacillus stearothermophilus NO2 was expressed in Saccharomyces cerevisiae 2805 under the adhl promoter. The CGTase was purified from S. cerevisiae 2805/pVT-CGTS. The purified enzyme exhibited a optima of activity around pH 7.0 and $65^{\circ}C$. Thermal stability of the enzyme was increased fairly as compared with the CGTase of B. stearothermophilus NO2. The conversion yield of cyclodextrin (CD) and the production ratio of $\alpha$-, $\beta$,-, ${\gamma}$-CD from starch were showed similarly aspect to the CGTase of B. stearothermophilus NO2.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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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.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.370-378
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• 1989

An Alkalophilic Bacillus circulans that can produce significant amount of cyclodextrin glucanotransferase (CGTase) was newly isolated from soil. The culture filtrate was successively purified by ($NH_4$)$_2$$SO_4$precipitation, DEAE-Sephadex column chromatography, and Sephadex G-100 column chromatography. The enzymatic properties, including molecular weight, optimal pH and temperature, stability, and kinetic parameters, were determined. The cyclodextrin synthesis reaction catalized by the purified CGTase was also studied. The sweet potato and corn starch were found to be the most suitable substrates with 60% conversion to cyclodextrin. The highest conversion was achieved at the CGTase concentration of 900-1,100 units/g of soluble starch. The purified CGTase could also catalize the transglycosylation on stevioside.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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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.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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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.

• 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%.

• KSBB Journal
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• v.9 no.5
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• pp.499-505
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• 1994

Bacillus macerans cyclodextrin glucanotransferase(EC 2.4.1.19: 1, 4-${\alpha}$-D(1, 4-${\alpha}$-glucano)-transferase, CGTase) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography. The molecular weight of the enzyme was 67,000, consisting of a subunit. The enzyme converted starch into ${\alpha}$-, ${\beta}$-, and ${\gamma}$-CD in the relative amounts of 1:1.68:0.32, respectively. In the early reaction period, maltohexose was formed mainly by the coupling reaction of ${\alpha}$-CD with D-glucose and then other oligosaccharides. Maltotetrose was formed mainly from ${\alpha}$-CD in the initial stage of hydrolysis of the enzyme and then small amount of other oligosaccharides. Maltotriose was a good substrate for the enzyme and maltosyl or D-glucopyranosyl group can be transfered from this sugar. In this work, D-glutosyl transfer was premiered.