• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.61-67
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• 1995

To investigate the role of induction on CGTase production for alkalophilic Bacillus firm us var. alkalophilus H609, the constitutive mutants that form a halo around its colonies at non-inducible AG agar media containing amylose and glucose were selected. The selected constitutive mutants could produce CGTase in the range of 18.9 to 28.8 units/ml $\cdot A_{600}$ in the alkaline basal medium, and finally a constitutive mutant Bacillus firmus var. alkalophilus CM46 was selected. The constitutive nature of CM46 was also confirmed in protein level using SDS-PAGE. The effects of induction and catabolite repression for both parent strain Bacillus firmus var. alkalophilus H609 and constitutive mutant CM46 were also compared by adding soluble starch and glucose during cultivation. The selected mutant CM46 was a non-inducible but a catabolite regulated type mutant. Even though inductive regulation was released, the specific CGTase activity defined as CGTase activity per cell concentration was not increased compared with that of parent strain. The cell growth and CGTase production patterns of constitutive mutant Bacillus firmus var. alkalophilus CM46 were compared with the parent strain to identify CGTase production characteristics.

• 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.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.63-69
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• 1991

Performance of column type bioreactor packed with immobilized cyclodextrin glucanotransferase (CGTase) on chitosan and Amberite IRA 900 was evaluated for cyclodextrin(CD) production. For CGTase immobilized on chitosan, the maximum CD conversion yield of 42% was achieved at the range of 88-168 units of immobilizied CGTase per gram of chitosan, retention time of 0.3 hr, and from 5.0% (w/v) of partially cyclized soluble starch. On the other hand, for CGTase immobilized on Amberite IRA 900, the maximum conversion yield of 40% was obtained at the range of 3.6-11.0 units of immobilized CGTase per gram of carrier and retention time of 1.2 hr from 5.0% of substrate. Above CD conversion yields are almost identical level with that can be obtained with soluble CGTase of 47%. The productivities of bioreactor packed with immobilized CGTase were 17.0g of CD/lㆍhr for amberite IRA 900 and 15.5g of CD/lㆍhr for chitosan. The partially cyclized starch with soluble CGTase were more suitable as substrate to achieve better CD conversion yield, and 5% (w/v) of partially cyclized soluble starch containing 10% (w/w) of CD was found to be most suitable to obtain maximum CD conversion yield.

• KSBB Journal
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• v.15 no.1
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• pp.35-41
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• 2000

We tried to prepare encapsulated whole cell cyclodextrin glucanotransferase(CGTase) in order to produce glycosyl-xylitol using xylitol as glucosyl acceptor. The organic nitrogen source was more effective for the production of CGTase from Bacillus macerans IFO 3490 than the inorganic one. Most of the CGTase which had been produced during cultivation was excreted to the growth medium. B. macerans cells inocculated in the capsule failed to grow to the high cell density. Adsorbents such as activated charcoal, Sephadex and Amberite resins could not adsorb efficiently the CGTase from the broth solution. We obtained successfully the encapsulated whole cell CGTase by immobilizing the concentrated broth solution in the calcium alginate capsules. The encapsulated whole cell CGTase carried out the transglycosylation reaction which converts xylitol into glucosyl-xylitol using dextrin as glucosyl donor.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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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 Life Science
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• v.12 no.6
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• pp.688-693
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• 2002

Molecular chaperones prevent the misfolding of newly synthesized polypeptides in the cell. The coexpression of molecular chaperones could be expected to improve the production of soluble and active recombinant proteins. In this study, the effect of coexpression of E. coli GroEL/ES chaperone on the active production of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in E. coli was investigated. Two plasmids, pTCGT1 and pGro7 in which the cgt and the groEL/ES genes are under the control of 77 promoter and araB promoter, respectively, were co-transformed into E. coli. With a series of cultures of recombinant E. coli cells, the optimal concentrations of IPTG and L-arabinose were found be 1 mM and 0.3 mg/$m\ell$, respectively. When IPTG and L-arabinose were added at 0.8~1.0 $OD_{600}$ and 0.4~0.5 $OD_{600}$, active CGTase production was increased significantly. This coexpression condition resulted in 1.5-fold increased level of soluble CGTase (0.7~0.73 unit/$m\ell$), compared to the level of CGTase in the single expression (0.36~0.56 unit/$m\ell$). An SDS-PACE analysis revealed that about 33.6% of CGTase in the total CGTase protein was found in the soluble fraction by coexpression of GroEL/ES chaperone.

• Journal of Microbiology and Biotechnology
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• v.8 no.2
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• pp.152-157
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• 1998

To analyze the role of the C and D domains in the cyclization activity of cyclodextrin glucanotransferase (CGTase), two plasmids, pKB1ΔC300 and pKB1ΔD96, were constructed in which DNA regions encoding 100 and 32 amino acids, respectively, from the C and D domains of B. stearothermophilus NO2 CGTase were deleted. The mutated CGTase from the pKBlΔC300 produced much lower amounts of ${\alpha}$-, ${\beta}$-, and $\gamma$-cyclodextrin (CD) than the parental CGTase. However, the mutated CGTase from the pKBlΔD96 showed a similar production pattern of CDs to wild-type CGTase. The production ratios of the ${\alpha}$-, ${\beta}$- and $\gamma$-CDs were not affected by the deletions, when compared to those of parental CGTase. The optimum temperature of the mutated CGTase from the pKBlΔC300 was decreased from $60^{\circ}C$ to $55^{\circ}C$. The optimum pH of the mutated CGTase from the pKB1D96 was shifted from 6.0 to 7.0. The thermostability of the two mutant CGTases were not changed. From these results, it is suggested that the C and D domains are not related to cyclization activity directly because mutant-enzymes deleted C or D domains still possessed their activity. However, they are important for other enzymatic properties such as productivity and pH optimum as a partition of CGTase tertiary structure.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.416-424
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• 1995

Mechanism of the cyclodextrin (CD) production reaction by cyclodextrin glucanotransferase (CGTase) using swollen extrusion starch as substrate was investigated emphasizing the structural features of starch granule. The degree of gelatinization was identified to be the most representative structural characteristic of swollen starch. The most suitable degree of gelatinization of swollen starch for CD production was around 63.52%. The structural transformation of starch granule during enzyme reaction was also followed by measuring the changes of the degree of gelatinization, microcrystallinity, and accessible and inaccessible portion to CGTase action of residual swollen starch. The adsorption phenomenon of CGTase to swollen starch was also examined under various conditions. The inhibition mechanism of CGTase by various CDs was identified to be competitive, most severely by a-CD. The mechanism elucidated will be used for development of a kinetic model describes CD production reaction in heterogeneous enzyme reaction system utilizing swollen extrusion starch.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.344-347
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• 1992

Simultaneous liquefaction and cyclodextrin (CD) production were conducted on potato starch using cyclomaltodextrin glucanotransferase (CGTase) from a mutant strain MNNG 8 of Bacillus stearothermophilus No. 239. A high concentration (30%) of potato starch was converted to cyc1o-dextrins (CDs) with 29% yield in the conditions of pH 6.0, temperature $80^{\circ}C$, 4.3 mM $CaCl_2$, CGTase addition of 3.0 dextrinizing activity unit (DAU) at $40^{\circ}C$/g starch.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.171-178
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• 1991

The two-stage enzyme reactor, packed with cyclodextrin glucanotransferase (CGTase) immobilized on Amberite IRA 900, coupled with ultrafiltration membrane was investigated for continuous production of cyclodextrin (CD). 5% (w/v) of soluble starch was partially cyclized, in the 0.1 l first-stage immobilized enzyme reactor, up to CD conversion yield of 10% (w/w) at retention time of 0.56hr and 1.5 units of immobilized CGTase/1g of carrier. In the second stage main immobilized enzyme reactor capacity of 1.5 l, the maximum CD conversion yield of 39% (w/v) was achieved at retention time of 2.8hr and 0.47 unit of CGTase/1 g of carrier. Unreacted residual dextrin was fractionated with ultrafiltration membrane, and then, recycled into the second-stage main bioreactor to increase the CD conversion yield. The most suitable membrane size and the volume concentration ratio (concentrate: filterate) for recycling of unreacted residual dextrin were found to be 5K dalton and 4:6, respectively. CD conversion yield was increased about 3~4% upon co-immobilization of pulluanase along with CGTase. Spent Amberite IRA 900 can be reutilized consecutively more than 3 times for immobilization of CGTase after regeneration.