• 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.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.368-371
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• 1991

The influence of ethylene glycol, glycerol, sorbitol and sucrose on the thermostability of Bacilus stearothermophzlus cyclodextrin glucanotransferase (CGTase) was investigated. Glycerol, sorbitol and sucrose had effect on thermostability of the CGTase. The effects appeared to be strongly dependent on concentration of additives. The thermostability of CGTase also was assayed in organic solvents such as n-butanol, l, &dioxane, n-octane. The therrnostability of CGTase increased in l, 4-dioxane and n-octane. Particularly, in n-octane, the CGTase retained the 81% of the initial activity after incubation at $75^{\circ}C$ for 90 min.

• Journal of Life Science
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• v.14 no.3
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• pp.391-395
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• 2004

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

• 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 ${\alpha}-amylase$.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.313-314
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• 1991

- The cyclodextrin glucanotransferase (CGTase) of a mutant of Bacillus stearothermophilus was purified in one step by affinity chromatography. The recovery was 95%. The specific activity of the CGTase increased from 26.2 U/mg protein to 485.5 U/mg protein. The purified CGTase was almost homogeneous by SDS-polyacrylamide gel electrophoresis. The one-step purification proved to be feasible with the mutant in contrast to the parent strain, which required pre-purification step of ammonium sulfate precipitation.

• 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.

• 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.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.

• Journal of Life Science
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• v.14 no.1
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• pp.121-125
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• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.671-674
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• 2001

Cyclodextrin glucanotransferase(CGTase) derived from recombinant Bacillus subtilis was partial purified and concentrated by ultrafiltration. The prepared CGTase were immobilized on various matrices by ionic interaction or covalent bond. CGTase covalently bound on CNBr-activated sepharose 4B were identified to be the highest immobilization activity among various immobilization methods. The optimum conditions for CGTase immobilization were determined; $30^{\circ}C$, 6Orpm, using O.2g CNBr-activated sepharose 4B in pH 6.0 phosphate buffer and 9hr immobilization.