Preliminary Study of Reaction Conditions for the Synthesis of AA-2G by Cyclodextrin Glucosyltransferase

HE Yamei; LIU Zhenyang; ZHENG Wan; SU Ruiyang; WU Huawei

doi:10.13386/j.issn1002-0306.2022090140

Volume 44 Issue 15

Jul. 2023

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Science and Technology of Food Industry > 2023 > 44(15): 203-212. > DOI: 10.13386/j.issn1002-0306.2022090140

HE Yamei, LIU Zhenyang, ZHENG Wan, et al. Preliminary Study of Reaction Conditions for the Synthesis of AA-2G by Cyclodextrin Glucosyltransferase[J]. Science and Technology of Food Industry, 2023, 44(15): 203−212. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022090140.

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Preliminary Study of Reaction Conditions for the Synthesis of AA-2G by Cyclodextrin Glucosyltransferase

College of Life Sciences, Yangtze University, Jingzhou 434025, China

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Received Date: September 14, 2022
Available Online: June 05, 2023

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Abstract

To investigate the effect of cyclodextrin glucosyltransferase (CGTase) produced by the recombinant strain pET-28a(+)-cgt-T1/BL21 (DE3) on the synthesis of 2-O-α-D-glucoside (AA-2G), the recombinant protein CGTase-T1 was expressed in the fermentation medium LB, purified by affinity chromatography and concentrated, followed by the determination of β-cyclization and disproportionation activity. AA-2G was synthesized using vitamin C and β-cyclodextrin as substrates. The effects of different glycan donors, substrate concentration, pH, temperature, substrate ratio, protein concentration and reaction time on AA-2G yield were further explored by single-factor optimization experiments, and the kinetics of CGTase were also analyzed. The results showed that CGTase could synthesize AA-2G, and the yield of AA-2G was 0.67 g/L before optimization of reaction conditions. Considering low cost and economic benefits, soluble starch and maltodextrin were selected as sugar donors in reaction condition optimization. When soluble starch was used as glycan donor, the yield of AA-2G catalyzed by the recombinant CGTase could reach up to 12.68 g/L under the condition as follows: The concentration of soluble starch at 70 g/L, the amount of V_C: glycosyl at 3:3, CGTase-T1 used at 5.0 mg/mL, pH4.5 at 37 ℃ for 42 h reaction. While maltodextrin was used as glycan donor, the yield of AA-2G catalyzed by the recombinant CGTase could reach up to 4.96 g/L under the condition as follows: the concentration of soluble starch at 30 g/L, the amount of V_C: glycosyl at 4:2, CGTase-T1 used at 5.0 mg/mL, pH5.0 at 37 ℃ for 30 h reaction. Under the optimal conditions, the yield of AA-2G was 18.93 times and 7.40 times higher than those of the unoptimized reaction conditions respectively. Through the kinetic analysis of CGTase-T1, it was found that the catalytic efficiency of soluble starch as sugar donor was higher than that of maltodextrin. Therefore, soluble starch as sugar donor was better than maltodextrin, resulting in a higher yield of AA-2G synthesis. In this study, the recombinant CGTase-T1 was successfully applied in the synthesis of AA-2G. By optimizing the reaction conditions of enzymatic catalysis, the output of AA-2G was greatly increased, providing a reference for the industrial production of AA-2G.

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2.	李丽，赵红梅. 油茶果壳活性炭的制备及其对阿莫西林的吸附作用. 保山学院学报. 2024(02): 6-12 .
3.	杜国丰，陈小焕，梁飞龙，尹梦琪，杨杰书，梁海. 羧基化改性海带渣对结晶紫的吸附性能研究. 粮食与油脂. 2024(05): 84-90 .
4.	张帮程，王立. ZnCl_2活化废旧涤纶基活性炭的制备及吸附性能. 无机盐工业. 2024(07): 126-134 .
5.	梁文俊，卢丹，胡玮. 商用活性炭用于加油站油气典型VOCs吸附性能评价. 环境工程. 2024(10): 65-72 .