• 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.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.74-79
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• 2008

The purpose of this study is to clone cgt gene from Paenibacillus sp. JB-13 and to overexpress the protein in E. coli. For this purpose, the cgt gene was amplified from Paenibacillus sp. JB-13 genomic DNA by PCR using degenerate oligonucleotide primers. The sequence analysis results showed that the cgt gene from Paenibacillus sp. JB-13 has 98% homology with the cgt gene of Bacillus sp. To overexpress the protein, the cgt gene was cloned into pEXP7 expression vector and transformed into E. coli. The production of CGTase by recombinant E. coli was optimized under following conditions: 0.5% glucose, 3.0% polypeptone, 0.3% $K_2HPO_4$, 0.5% NaCl, and 7.0 of initial pH, 2.0% of inoculum, $37^{\circ}C$ of culture temperature for 14 hr. And the optimal agitation was found at 0.1 vvm. The synthesis of 2-O-${\alpha}$-D-Glucopyranosyl L-Ascorbic acid (AA-2G) using the CGTase expressed in E. coli was identified as AA-2G by HPLC and HPLC confirmed that treating AA-2G made by cloned CGTase with ${\alpha}$-glucosidase substantially produced AA and glucose.

• Microbiology and Biotechnology Letters
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• v.48 no.1
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• pp.12-23
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• 2020

Edible films containing antimicrobial agents can be used as safe alternatives to preserve food products. Essential oils are well-recognized antimicrobials. However, their low water solubility, volatility and high sensitivity to oxygen and light limit their application in food preservation. These limitations could be overcome by embedding these essential oils in complexed product matrices exploiting the encapsulation efficiency of β-cyclodextrin. This study focused on the maximization of β-cyclodextrin production using cyclodextrin glucanotransferase (CGTase) and the evaluation of its encapsulation efficacy to fabricate edible antimicrobial films. Response surface methodology (RSM) was used to optimize CGTase production by Brevibacillus brevis AMI-2 isolated from mangrove sediments. This enzyme was partially purified using a starch adsorption method and entrapped in calcium alginate. Cyclodextrin produced by the immobilized enzyme was then confirmed using high performance thin layer chromatography, and its encapsulation efficiency was investigated. The clove oil/β-cyclodextrin inclusion complexes were prepared using the coprecipitation method, and incorporated into chitosan films, and subjected to antimicrobial testing. Results revealed that β-cyclodextrin was produced as a major product of the enzymatic reaction. In addition, the incorporation of clove oil/β-cyclodextrin inclusion complexes significantly increased the antimicrobial activity of chitosan films against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Escherichia coli, and Candida albicans. In conclusion, B. brevis AMI-2 is a promising source for CGTase to synthesize β-cyclodextrin with considerable encapsulation efficiency. Further, the obtained results suggest that chitosan films containing clove oils encapsulated in β-cyclodextrin could serve as edible antimicrobial food-packaging materials to combat microbial contamination.

• KSBB Journal
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• v.19 no.2
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• pp.164-167
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• 2004

Cyclodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) is one of the most applied industrial enzymes that produces cyclodextrins from starch and related ${\alpha}$-1,4-glucans by intramolecular transglycosylation reaction upon Ca$\^$2＋/ dependent manner. The reaction of CLEC, ${\alpha}$-CGTases from Bacillus macerans with the soluble starch as a substrate reveals that the surfactants (SDS, N-octyl-${\beta}$-D-glucoside) significantly affect not only the overall products of CDs but also their selectivity. The surfactants (SDS, Lubrol PX) trigger the increase of ${\alpha}$-CD production, but Triton x-100 and Tween 80 suppress ${\alpha}$-CD specificity. Organic solvents (dimethyl sulfoxide, formamide, 2-methyl-2,4-pentandiol, and ethylene glycol) also cause changes of total product and product selectivity.

• 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.11 no.2
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• pp.242-250
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• 2001

A Gram-positive bacterium (strain JB-13) that was isolated from soil as a producer of cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] was identified as Panibacillus sp. JB-13. This CGTase could catalyze the transglucosylation reaction from soluble starch to L-ascorbic acid (AA). A main product formed by this enzyme with ${\alpha}-glucosidase$ was identified as $2-O-{\alpha}-D-glucopyranosyl{\;}_{L}-ascorbic$ acid (AA-2G) by the HPLC profile and the elemental analysis. CGTase was purified to homogeneity using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Seohadex A-50, and gel chromatography on Sephacryl S-200HR. The molecular weight was determined to be 66,000 by both gel chromatography and SDS-PAGE. The isoelectric point of the purified enzyme was 5.3. The optimum pH and temperature was PH 7.0 and $45^{\circ}C$ respectively. The enzyme was stable in the range of pH 6-9 and at temperatures of $75{\circ}C$ or less in the presence of 15 mM ${CaCl_2}.\;{Hg^2+},\;{Mn^+2},{Ag^+},\;and\;{Cu^2+}$ all strongly inhibited the enzyme's activity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.78-81
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• 1998

Cyclodextrin glucanotransferase [CGTase, E.C.2.4.1.19] is an extracellular enzyme, which catalyzes he formation of ${\alpha}$-, ${\beta}$-, ${\gamma}$- CDs from starch. Their proportions of formations depend on enzyme sources and reaction conditions. To understand what determines the product specificity of CGTases, we examined the alteration of product specificity of CGTase from Bacillus macerans by organic solvent sand pH. At acidic pH range less than pH 6 where the enzyme was unstable, the ratio of ${\alpha}$-/ ${\beta}$-CD production was increased 4 times more than that at neutral pH range. As we increased the concentration of 2-butanol, ${\alpha}$-/ ${\beta}$-CD ratio was proportionally increased but / ratio remained constant. The ${\alpha}$-/ ${\beta}$-CD ratio of products was increased in the reaction media which yielded low products.

• The Korean Journal of Food And Nutrition
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• v.8 no.3
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• pp.253-261
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• 1995

A strain of alkalophilic Bacillus sp. C-21 has been Isolated from sold. The strain was capable of producing large amount of cyclodextrin glycosyltransferase (CGTase) in the high alkaline pH medium. The preferable medium composition was determined to be as follows : 1.0% soluble starch, 1.0% peptone, 0.5% yeast extract, 0.1% K2HP04, 0.02% MgSO4.7H2O and 1.0% Na2CO3(pH 10.0) The highest enzyme production was observed after 30hours of cultivation at 33$^{\circ}C$. The optimum temperature and pH for the activity of crude enzyme were 6$0^{\circ}C$ and 6.0, respectively. The enzyme was stable between pH 6.0 and 9.6, and up to 55$^{\circ}C$.

• KSBB Journal
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• v.19 no.2
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• pp.159-163
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• 2004

The X-ray structure of the cydodextrin-glucanotransferase of Bacillus macerans was solved by molecular replacement at 2.0 ${\AA}$ resolution. The refined structure has a crystallographic R-factor of 16.6%, (R$\sub$free/ = 20.5%). A new metal binding site occupied by two Ca$\^$2+/-ions was found at an accession channel of the active site. There is a large accumulation of negative charges that bind these Ca$\^$2+/-ions, thereby connecting segment ${\beta}$13-${\alpha}$G (residue 254-276) to the main body of domain A (at ${\alpha}$H, residue 283-297). The segment 313-${\alpha}$G contains the catalytic residue Glu258 between subsite 1 and -1 and Tyr260 (subsite 2) which is located at the entrance of the active site. The Ca$\^$2＋/-site 3a,b may have a major role for the activity and specificity of this CGTase, although it is not even conserved for the a-subclass of CGTases.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.31-36
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• 2001

Paenibacillus sp. JB-13 producing the cyclodextrin glucan-otransferase(CGTase) [EC 2.4.1.19] that glucosylated ascorbic acid(AA) at the C-2 position was isolated form soil and the optimal conditions for the production of 2-O-$\alpha$-D- Glucopyranosl L-Ascorbic acid(AA-2G) with CGTase were investigated. CGTase produced AA-2G efficiently using dextrin as a substrate and AA as an aceptor. Several AA-2-oilgosaccharides(AA-2Gs) were also produced in this reaction mixture, and these were efficiently hydro-lyzed to AA-2G and glucose by the treatment with glucoamylase. The optimal temperature for AA-2G production was $37^{\circ}C$ and the optimal pH was around 6.5. CGTase also utilized $\alpha$-,$\beta$-,${\gamma}$-CDs, soluble starch, com statch, dia-static solution from rice and diastatic solution from malt as substrate, but not glucose. The reaction mixture for the maximal production of AA-2G was following; 15% total substrate concentration, 2,500 units/ml of CGTase and a mixing ration of 3:2(g of AA: g of dextrin). Under this condition, 56 mM of AA-2G ,which corresponded to 12.4% yield based on AA. was produced after incubation for 44 hrs at $37^{\circ}C$ and pH 6.5.