• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.1
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• pp.49-56
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• 2000

The 2-O-$\alpha$-D-glucopyranosyl L-ascorbic acid (AA-2G) which was enzymatically glucosylated with the cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] from Bacillus sp. JK-43 has been reported previously. The presnet experiments examined the optimal conditons for the productio of AA-2G from AA and soluble starch, and characterized the properties of the CGTase from Bacillus sp. JK-43. The reaction mixture for the maximal production of AA-2G was followings; 12% total substrate concentration, 1,400 usits/mL of CGTase and a mixing ratio of 2 : 3(g or AA : g of soluble starch). Under this condition, 1.76mM of AA-2G, which corresponded to 2.53% yield based on AA, was produced after incubation for 24hrs at 45$^{\circ}C$ (pH 5.5). The optimum pH and temperature for the CGTase activity were 6.0 and 45$^{\circ}C$, respectively. The enzyme was stable at pH 5.5 to 9.5, and at temperature up to 5$0^{\circ}C$. The thermostability of the enzyme could be enhanced up to 6$0^{\circ}C$ by the addition of 30mM CaCl2.

• Applied Biological Chemistry
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• v.47 no.2
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• pp.187-191
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• 2004

For the production of $2-O-{\alpha}-D-glucopyranosyl-L-ascorbic$ acid (ascorbic acid-2-g1ucoside, AA-2G) from ascorbic acid, the usability of spinach seed as the source of ${\alpha}-glucosidase$ having transglucosylation activity was studied. The optimum conditions for the production of AA-2G from ascorbic acid and glucose donor were investigated by using crude extract of Spinachia oleracea L. Woosung, the selected source of enzyme. The production of AA-2G was the highest with 1.053 mM when spinach seeds were grown for 2 days after germination. Maltose was the most effective glucose donor, and the optimum concentration of ascorbic acid and maltose were 175 mM and 225 mM, respectively. The optimum concentration of ${\alpha}-glucosidase$ was 60 units. The most effective buffer was sodium acetate and its optimum concentration was 175 mM. The optimum pH and temperature were 5.0 and $65^{\circ}C$, respectively. Under the optimum condition, 2.14 mM of AA-2G was produced from ascorbic acid after 50 minutes of reaction.

• 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.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.368-376
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• 2003

Cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. JB-13 was immobilized on various carriers by several immobilization methods such as ionic binding, covalent linkage and ultrafiltration to improve the process performance. The ultrafiltration and covalent linkage with CNBr-activated sepharose 4B were found as the best method for immobilization of CGTase. The ability of CGTase immobilization onto CNBr-activated sepharose 4B was as high as 18,000 units/g resin when the conditions was as follows: contact time 9 hrs at $37^{\circ}C$, pH 6.0, 100 nm and enzyme loading 24,000 units/g resin. The optimum conditions for production of 2-O-$\alpha$-D-Glucopyranosyl L-Ascorbic acid by immobilized CGTase turned out to be: pH 5.0, temperature $37^{\circ}C$, 20% substrate solution containing 8% (w/v) of soluble starch and 12% (w/v) of L-ascorbic acid sodium salt, 100 rpm, far 25 hrs and with 800 units of immobilized CGTase/ml substrate solution. Moreover the CGTase activity could be stably maintained for 8 times of repetitive reactions after removing products by ultrafiltration through YM 10 membrane.

• Applied Biological Chemistry
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• v.43 no.1
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• pp.12-17
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• 2000

For the enzymatic production of $2-O-{\alpha}-D-glucopyranosyl-L-ascorbic$ acid (AA-2G) from ascorbic acid, rice seed was used as the source of ${\alpha}-glucosidase$ having transglucosylation activity. Among six rice varieties, cultivated in Korea, ${\alpha}-glucosidase$ activity of Oryza savita L. cv. Ilpumbyeo was the highest with 125.03 unit/ml and it had maximum specific activity with 8.52 unit/mg protein when rice seeds were grown for 3 days after germination. For the production of AA-2G using crude extract of O. savita L. cv. Ilpumbyeo, maltose was most effective glucose donor. The optimum concentration of maltose and ascorbic acid were 125 mM and 175 mM, respectively. The optimum concentration of ${\alpha}-glucosidase$ was 100 unit. The most effective buffer was 100 mM sodium citrate. The optimum pH and temperature were 5.0 and $60^{\circ}C$, respectively. Under the optimum condition, $108.43\;{\mu}M/unit$ of AA-2G was produced from ascorbic acid after 35 minutes of reaction, which corresponds to 6.2% of conversion ratio based on the amount of ascorbic acid used.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.1 s.49
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• pp.111-114
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• 2005

This study is about the cosmeceutical products using enzyme induced bio-conversion system. In general, ascorbic acid (AA) has the higher reducing activity and can be used for various purpose in the cosmetics. But it is very unstable in the aqueous system and difficult to maintain its stability in the cosmetics product. 2-O-$\alpha$-D-Glucopyranosyl-L-ascorbic acid (AA2G) is the stabilized form of AA and showed the less whitening activity than AA. In this study, we developed bio-conversion system improving the stability and efficacy of AA2G and AA, respectively. In this system, AA2G (over $80\%$) can be converted to AA and glucose within 30 min. The converted product showed higher anti-tyrosinase activity like AA (AA2G showed no anti tyrosinase activity) and depigmenting activity in the artificial tanning test. From these results, we could conclude this system is a brand new method to increase the activity of AA and maintain its stability.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.4
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• pp.609-617
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• 1998

A microorganism capable of producing high level of extracellular cyclodextrin glucanotransferase(EC 2.4.1.19 ; CGTase) was isolated from Kimchi. 2-O-$\alpha$-D-glucopyranosyl L-ascorbic acid(AA-2G) was synthesized by transglycosylation reaction of CGTase using starch as a donor and L-ascorbic acid as an acceptor. The isolated strain S-6 was identified as Bacillus sp. S-6. The maximal CGTase production was observed in a medium containing 0.5% soluble starch, 1% yeast extract, 1% NaCO3, 0.1% K2HPO4, and 0.02% MgSO4 with initial pH 8.0. The strain was cultured at 37$^{\circ}C$ for 40 hr with reciprocal shaking. Using the culture supernatant as crude enzyme, the optimal pH and temperature of the CGTase activity of this strain were 7.0 and 4$0^{\circ}C$. In the effects of pH and temperature on the stability of the enzyme, the enzyme was stable in the range of pH 6.0~10.0 and up to 45$^{\circ}C$, respectively.

• Proceedings of the Korean Society of Life Science Conference
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• 2002.03a
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• pp.82-82
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• 2002

• Proceedings of the Korean Society of Life Science Conference
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• 2001.09a
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• pp.56-56
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• 2001

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.1
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• pp.41-48
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• 2000

A bacterial strain, designated as JK-43, producing extracellular cyclodextrin glucanotransferase (CGTase)[EC 2.4.1.19] was isolated from kimchi. The CGTase from isolated strain JK-43 showed the transglucosylation activity from soluble starch to L-ascorbic acid(AA) compared to those obtained from other strains. A main product formed by this reaction was identified as $2-O-{\alpha}-glucopyranosyl$ L-ascorbic acid(AA-2G) by testing its susceptibility to ${\alpha}-glucosidase$ hydrolysis, the HPLC profiles, and through the elementary analysis. the ${\beta}-CD,\;{\gamma}-CD$, potato starch and corn starch were identified to be suitable glucosyl donor for transglucosylation reaction on AA by CGTase. Acceptor specificity on AA-2G production was examined by use of AA, Iso-AA and AA-2P. Transglucosylation was observed toward AA-2P as well as AA and Iso-AA. The microorganism isolated from kimchi was identified as a strain of Bacillus sp. JK-43 based on the morphological, cultural, biochemical characteristics and partial 16SrDNA sequence analysis. The maximal CGTase production was observed in a medium containing 1.0% soluble starch, 1.0% yeast extract, 1.0% $Na_2CO_3\;0.1%\;K_2HPO_4,\;and\;0.02%\;MgSO_4{\cdot}7H_2O$ with initial pH 7.0. The strain was cultured at $37^{\circ}C$ for 26 hrs with reciprocal shaking.