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

Turn off MathJax

Article Contents

Abstract

References

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.

Citation:

PDF (2030 KB)

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

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

More Information

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

Graphical Abstract

Abstract

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.

FullText(HTML)

References (50)

References

[1]	苏桂棋, 黄和林, 蒋娜, 等. 维生素C的作用及常见不良反应[J]. 世界最新医学信息文摘,2019,19(8):120−121, 125. [SU G Q, HUANG H L, JIANG N, et al. The role of vitamin C and common adverse reactions[J]. World Latest Medical Information Digest,2019,19(8):120−121, 125. doi: 10.19613/j.cnki.1671-3141.2019.08.056 SU G Q, HUANG H L, JIANG N, et al. The role of vitamin C and common adverse reactions[J]. World Latest Medical Information Digest, 2019, 19(8): 120-121, 125. doi: 10.19613/j.cnki.1671-3141.2019.08.056
[2]	NAIDU K A. Vitamin C in human health and disease is still a mystery? An overview[J]. Nutrition Journal,2003,2(1):7. doi: 10.1186/1475-2891-2-7
[3]	饶建华, 韩璐, 张自萍. 维生素C糖苷类衍生物的研究进展[J]. 中国新药杂志,2012,21(7):761−766. [RAO J H, HAN L, ZHANG Z P. Research progress of vitamin C glycoside derivatives[J]. China Journal of New Drugs,2012,21(7):761−766. RAO J H, HAN L, ZHANG Z P. Research progress of vitamin C glycoside derivatives[J]. China Journal of New Drugs, 2012, 21(7): 761-766.
[4]	HAN R, LIU L, LI J, et al. Functions, applications and production of 2-O-D-glucopyranosyl-L-ascorbic acid[J]. Applied Microbiology and Biotechnology,2012,95(2):313−320. doi: 10.1007/s00253-012-4150-9
[5]	李标, 唐双焱. 2-O-α-D-吡喃葡萄糖基抗坏血酸研究进展[J]. 广西科学,2017,24(1):48−53. [LI B, TANG S Y. Research progress of 2-O-α-D-glucopyranosyl-ascorbic acid[J]. Guangxi Science,2017,24(1):48−53. LI B, TANG S Y. Research progress of 2-O-α-D-glucopyranosyl-ascorbic acid[J]. Guangxi Science, 2017, 24(1): 48-53.
[6]	WAKAMIYA H, SUZUKI E, YAMAMOTO I, et al. Vitamin C activity of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid in guinea pigs[J]. Journal of Nutritional Science & Vitaminology,1992,38(3):235−245.
[7]	ZHANG W, HUANG Q, YANG R, et al. 2-O-D-glucopyranosyl-L-ascorbic acid: Properties, production, and potential application as a substitute for L-ascorbic acid[J]. Journal of Functional Foods,2021,82(7):104481.
[8]	TOYODAONO Y, MAEDA M, NAKAO M, et al. 2-O-(beta-D-glucopyranosyl) ascorbic acid, a novel ascorbic acid analogue isolated from Lycium fruit[J]. Journal of Agricultural and Food Chemistry,2004,52(7):2092−2096. doi: 10.1021/jf035445w
[9]	YAMAMOTO I, MUTO N, NAGATA E, et al. Formation of a stable L-ascorbic acid alpha-glucoside by mammalian alpha-glucosidase-catalyzed transglucosylation[J]. Biochimica et Biophysica Acta,1990,1035(10):44−50.
[10]	TANAKA M. Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation[J]. Biochimica et Biophysica Acta,1991,1078(2):127−132. doi: 10.1016/0167-4838(91)99000-1
[11]	LEE S, LIM J, LEE J H, et al. Ascorbic acid 2-glucoside stably promotes the primitiveness of embryonic and mesenchymal stem cells through ten-eleven translocation and cAMP-responsive element-binding protein-1-dependent mechanisms[J]. Antioxidants & Redox Signaling,2004,32(1):35−59.
[12]	GUDIMINCHI R K, NIDETZKY P. Walking a fine line with sucrose phosphorylase: efficient single-step biocatalytic production of 1-ascorbic acid 2-glucoside from sucrose[J]. Chembiochem:a European Journal of Chemical Biology,2017,18(14):1387−1390. doi: 10.1002/cbic.201700215
[13]	MUKAI K, TSUSAKI K, KUBOTA M, et al. Process for producing 2-O-alpha-D-glucopyranosyl-L-ascorbic acid: Europe, EP20030741243[P]. 2003-07-07.
[14]	黄敏, 裘娟萍. 生物转化法生产2-O-α-D-吡喃型葡萄糖基L-抗坏血酸的研究进展[J]. 工业微生物,2004(2):41−44. [HUANG M, QIU J P. Advances in research of preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid by biological transformation[J]. Industrial Microbiology,2004(2):41−44. doi: 10.3969/j.issn.1001-6678.2004.02.010 HUANG M, QIU J P. Advances in research of preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid by biological transformation[J]. Industrial Microbiology, 2004, (2): 41-44. doi: 10.3969/j.issn.1001-6678.2004.02.010
[15]	ZHANG Z, LI J, LIU L, et al. Enzymatic transformation of 2-O-α-D-glucopyranosyl-L-ascorbic acid by α-cyclodextrin glucanotransferase from recombinant Escherichia coli[J]. Biotechnology & Bioprocess Engineering,2011,16(1):107−113.
[16]	TAO X, SU L, WU J. Current studies on the enzymatic preparation 2-O-α-D-glucopyranosyl-L-ascorbic acid with cyclodextrin glycosyltransferase[J]. Critical Reviews in Biotechnology,2018,39(2):1−9.
[17]	陶秀梅. Bacillus stearothermophilus NO2环糊精葡萄糖基转移酶的分子改造及制备AA-2G的研究[D]. 无锡: 江南大学, 2020. TAO X M. Molecular modification of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase and research on the preparation of AA-2G[D]. Wuxi: Jiangnan University, 2020.
[18]	李晓涵, 郝建华, 郭姣梅, 等. 环糊精葡萄糖基转移酶高效异源表达研究进展[J]. 微生物学通报,2020,47(2):615−622. [LI X H, HAO J H, GUO J M, et al. Advance in high-level heterologous expression of cyclodextrin glucosyltransferase[J]. Bulletin of Microbiology,2020,47(2):615−622. doi: 10.13344/j.microbiol.china.190295 LI X H, HAO J H, GUO J M, et al. Advance in high-level heterologous expression of cyclodextrin glucosyltransferase[J]. Bulletin of Microbiology, 2020, 47(2): 615-622. doi: 10.13344/j.microbiol.china.190295
[19]	余磊, 李骥璇, 王忆茗, 等. 蔗糖磷酸化酶全细胞催化AA-2G的条件优化[J]. 现代生物医学进展,2017,17(14):2601−2605. [YU L, LI J X, WANG Y M, et al. Optimization of whole-cell synthesis of AA-2G by sucrose phosphorylase[J]. Advances in Modern Biomedicine,2017,17(14):2601−2605. doi: 10.13241/j.cnki.pmb.2017.14.001 YU L, LI J X, WANG Y M, et al. Optimization of whole-cell synthesis of AA-2G by sucrose phosphorylase[J]. Advances in Modern Biomedicine, 2017, 17(14): 2601-2605. doi: 10.13241/j.cnki.pmb.2017.14.001
[20]	郭姣梅. 海洋环糊精葡萄糖基转移酶的固定化及其性能研究[D]. 上海: 上海海洋大学, 2020. GUO J M. Study on immobilization of marine cyclodextrin glucosyltransferase and its properties[D]. Shanghai: Shanghai Ocean University, 2020.
[21]	郑丹妮. Bacillus sp. FJAT-44876 γ-环糊精葡萄糖基转移酶的表征及热稳定性提升研究[D]. 无锡: 江南大学, 2020. ZHENG D N. Characterization and thermostability improved of γ-CGTase from Bacillus sp. FJAT-44876[D]. Wuxi: Jiangnan University, 2020.
[22]	王蕾. 环糊精葡萄糖基转移酶的产物特异性分子改造及发酵制备研究[D]. 无锡: 江南大学, 2018. WANG L. Product specificity engineering and fermentation of cyclodextrin glycosyltransferase[D]. Wuxi: Jiangnan University, 2018.
[23]	李晓涵, 郭姣梅, 宋凯等. Bacillus sp. Y112环糊精葡萄糖基转移酶位点R81定点突变提高产物特异性[J]. 食品科学,2021,42(10):133−138. [LI X H, GUO J M, SONG K, et al. Improvement of the product specificity of Bacillus sp. Y112 cyclodextrin glucosyltransferase by site-directed mutagenesis of arginine 81[J]. Food Science,2021,42(10):133−138. doi: 10.7506/spkx1002-6630-20200205-031 LI X H, GUO J M, SONG K, et al. Improvement of the product specificity of Bacillus sp. Y112 cyclodextrin glucosyltransferase by site-directed mutagenesis of arginine 81[J]. Food Science, 2021, 42(10): 133-138. doi: 10.7506/spkx1002-6630-20200205-031
[24]	左方圆. Bacillus stearothermophilus NO2环糊精葡萄糖基转移酶的分子改造及制备α-环糊精的研究[D]. 无锡: 江南大学, 2022. ZUO F Y. Molecular modification of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase and preparation of α-cyclodextrin[D]. Wuxi: Jiangnan University, 2022.
[25]	柴宝成. 分子改造环糊精葡萄糖基转移酶合成糖基化染料木素[D]. 无锡: 江南大学, 2021. CHAI B C. Engineering of cyclodextrin glucosyltransferase for the synthesis of glycosylated genistein[D]. Wuxi: Jiangnan University, 2021.
[26]	花敬涵. β-环糊精糖基转移酶的催化机制及理性改造研究[D]. 合肥: 合肥工业大学, 2019. HUA J H. Catalytic mechanism and rational transformation of β-cyclodextrin glycosyltransferase[D]. Hefei: Hefei University of Technology, 2019.
[27]	VIOLAINE, GERARD, EMEL, et al. Ascorbic acid derivatives as potential substitutes for ascorbic acid to reduce color degradation of drinks containing ascorbic acid and anthocyanins from natural extracts.[J]. Journal of Agricultural and Food Chemistry,2019,67(43):12061−12071. doi: 10.1021/acs.jafc.9b05049
[28]	AGA H, YONEYAMA M, SAKAI S, et al. Synthesis of 2-O-α-D-glucopyranosyl 1-ascorbic acid by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus[J]. Journal of the Agricultural Chemical Society of Japan,1991,55(7):1751−17556.
[29]	LIU Z, WU G, WU H. Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1[J]. 3 Biotech,2022,12(3):58. doi: 10.1007/s13205-022-03111-8
[30]	BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical biochemistry,1976,72:248−254. doi: 10.1016/0003-2697(76)90527-3
[31]	TESFAI B T, WU D, CHEN S, et al. Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii[J]. Journal of Inclusion Phenomena & Macrocyclic Chemistry,2013,77(1-4):147−153.
[32]	VANDERVEEN B A, VANALEBEEK G J, UITDEHAAG J C, et al. The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms[J]. European Journal of Biochemistry,2001,267(3):658−665.
[33]	韩瑞枝. 环糊精葡萄糖基转移酶的分子改造及合成糖基化L-抗坏血酸[D]. 无锡: 江南大学, 2013. HAN R Z. Molecular modification of cyclodextrin glucosyltransferase and synthesis of glycosylated L-ascorbic acid[D]. Wuxi: Jiangnan University, 2013.
[34]	宋凯. 海洋环糊精葡萄糖基转移酶的表达、酶学性质及AA-2G制备的研究[D]. 上海: 上海海洋大学, 2021. SONG K. Heterogeneous expression, enzyme properties and preparation of AA-2G of cyclodextrin glycosyltransferase[D]. Shanghai: Shanghai Ocean University, 2021.
[35]	张国超, 王岁楼, 吴晓宗. 环糊精葡萄糖转移酶的研究进展[J]. 生物学杂志,2008(2):55−58. [ZHANG G C, WANG S L, WU X Z. Research progress on cyclodextrin glucotransferase[J]. Journal of Biology,2008(2):55−58. doi: 10.3969/j.issn.2095-1736.2008.02.016 ZHANG G C, WANG S L, WU X Z. Research progress on cyclodextrin glucotransferase[J]. Journal of Biology, 2008(2): 55-58. doi: 10.3969/j.issn.2095-1736.2008.02.016
[36]	王月明, 孙万刚, 张斌等. 环糊精及环糊精葡萄糖基转移酶特性研究[J]. 安徽农业科学,2007(27):8430−8431, 8434. [WANG Y M, SUN W G, ZHANG B, et al. Study on the characteristics of cyclodextrin and cyclodextrin glucosyltransferase[J]. Anhui Agricultural Science,2007(27):8430−8431, 8434. doi: 10.3969/j.issn.0517-6611.2007.27.004 WANG Y M, SUN W G, ZHANG B, et al. Study on the characteristics of cyclodextrin and cyclodextrin glucosyltransferase[J]. Anhui Agricultural Science, 2007(27): 8430-8431+8434. doi: 10.3969/j.issn.0517-6611.2007.27.004
[37]	张国宁, 冯婧娴, 杨颖博等. 环糊精葡萄糖基转移酶在天然产物糖基化修饰中的应用[J]. 生物技术通报,2022,38(3):246−255. [ZHANG G N, FENG J X, YANG Y B, et al. Application of cyclodextrin glucosyltransferase in the glycosylation modification of natural products[J]. Biotechnology Bulletin,2022,38(3):246−255. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0642 ZHANG G N, FENG J X, YANG Y B, et al. Application of cyclodextrin glucosyltransferase in the glycosylation modification of natural products[J]. Biotechnology Bulletin, 2022, 38(03): 246-255. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0642
[38]	曹新志, 刘芳, 明红梅等. 酶法合成葡萄糖基-L-抗坏血酸的产酶条件及转化条件优化[J]. 农产品加工(学刊),2008(1):19−22, 28. [CAO X Z, LIU F, MING H M, et al. Optimization of enzyme production and transformation conditions for enzymatic synthesis of glucosyl-L-ascorbic acid[J]. Agricultural Products Processing (Journal),2008(1):19−22, 28. CAO X Z, LIU F, MING H M, et al. Optimization of enzyme production and transformation conditions for enzymatic synthesis of glucosyl-L-ascorbic acid [J]. Agricultural Products Processing (Journal), 2008(1): 19-22, 28.
[39]	HONG K J, KYUNG M B, SUNG K K. Production of 2-O-α-D-glucopyranosyl-L-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.[J]. Biotechnology Letters,2001,23(21):1793−1797. doi: 10.1023/A:1012413301061
[40]	EIBAID A, MIAO M, BASHARI M, et al. Biosynthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid from maltodextrin catalyzed by cyclodextrin glucanotransferase from Bacillus sp. SK13.002[J]. Journal of Food and Nutrition Research,2014,2(4):193−197. doi: 10.12691/jfnr-2-4-10
[41]	黄立萍, 郝建华, 王伟, 等. 2-氧-α-D-葡萄糖基-L-抗坏血酸酶法合成工艺优化[J]. 食品与机械,2018,34(11):7. [HUANG L P, HAO J H, WANG W, et al. Optimization of enzymatic synthesis of 2-O-α-D-glucosyl-L-ascorbate acid[J]. Food and Machinery,2018,34(11):7. HUANG L P, HAO J H, WANG W, et al. Optimization of enzymatic synthesis of 2-O-α-D-glucosyl-L-ascorbate acid[J]. Food and Machinery, 2018, 34(11): 7.
[42]	邢琳, 张秀华, 钊倩倩, 等. α-环糊精葡萄糖基转移酶的高效表达及酶法制备AA-2G条件优化[J]. 中国生化药物杂志,2016,36(11):5−8. [XING L, ZHANG X H, ZHAO Q Q, et al. High-efficiency expression of α-cyclodextrin glucosyltransferase and optimization of conditions for enzymatic preparation of AA-2G[J]. China Biochemical Medicine,2016,36(11):5−8. XING L, ZHANG X H, ZHAO Q Q, et al. High-efficiency expression of α-cyclodextrin glucosyltransferase and optimization of conditions for enzymatic preparation of AA-2G[J]. China Biochemical Medicine, 2016, 36(11): 5-8 .
[43]	CHEN S, XIONG Y, SU L, et al. Position 228 in Paenibacillus macerans cyclodextrin glycosyltransferase is critical for 2-O-D-glucopyranosyl-L-ascorbic acid synthesis[J]. Journal of Biotechnology, 2017, 247.
[44]	GUDIMINCHI R K, TOWNS A, VARALWAR S, et al. Enhanced synthesis of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid from alpha-cyclodextrin by a highly disproportionating CGTase[J]. Acs Catalysis,2016,6(3):1606−1615. doi: 10.1021/acscatal.5b02108
[45]	许乔艳, 韩瑞枝, 李江华等. 亚位点+1处突变提高软化类芽胞杆菌环糊精糖基转移酶底物麦芽糊精特异性[J]. 生物工程学报,2014,30(1):98−108. [XU Q Y, HAN R Z, LI J H, et al. Mutation at subsite+1 improves the specificity of maltodextrin as a substrate of cyclodextrin glycosyltransferase from Paenibacillus softening[J]. Chinese Journal of Biological Engineering,2014,30(1):98−108. doi: 10.13345/j.cjb.130401 XU Q Y, HAN R Z, LI J H, et al. Mutation at subsite+1 improves the specificity of maltodextrin as a substrate of cyclodextrin glycosyltransferase from Paenibacillus softening[J]. Chinese Journal of Biological Engineering, 2014, 30(1): 98-108. doi: 10.13345/j.cjb.130401
[46]	张兴荣, 李峰, 贺连智等. 高产β-环糊精葡萄糖基转移酶菌株的筛选、产酶条件优化及酶学性质研究[J]. 中国酿造,2020,39(11):85−91. [ZHANG X R, LI F, HE L Z, et al. Screening and enzyme production condition optimization of high-yield β-CGTase strain and enzymatic property[J]. China Brewing,2020,39(11):85−91. doi: 10.11882/j.issn.0254-5071.2020.11.017 ZHANG X R, LI F, HE L Z, et al. Screening and enzyme production condition optimization of high-yield β-CGTase strain and enzymatic property[J]. China Brewing, 2020, 39 (11): 85-91. doi: 10.11882/j.issn.0254-5071.2020.11.017
[47]	丛远华, 朱沁, 冯春波. 高含量烟酰胺与AA-2G复配体系的水溶液的稳定性研究[J]. 化学与生物工程,2019,36(11):52−57, 63. [CONG Y H, ZHU Q, FENG C B. Stability of aqueous solution of high content nicotinamide and AA2G mixed system[J]. Chemical and Biological Engineering,2019,36(11):52−57, 63. CONG Y H, ZHU Q, FENG C B. Stability of aqueous solution of high content nicotinamide and AA2G mixed system[J] Chemical and Biological Engineering, 2019, 36 (11): 52-57, 63.
[48]	单丽媛, 刘龙, 李江华等. 利用环糊精糖基转移酶转化合成AA-2G的研究[J]. 食品与生物技术学报,2018,37(1):27−32. [SHAN L Y, LIU L, LI J H, et al. Enzyme synthesis of AA-2G by cyclodextrin glycosyltransferase[J]. Journal of Food Science and Biotechnology,2018,37(1):27−32. doi: 10.3969/j.issn.1673-1689.2018.01.005 SHAN L Y, LIU L, LI J H, et al. Enzyme synthesis of AA-2G by cyclodextrin glycosyltransferase[J]. Journal of Food Science and Biotechnology, 2018, 37(1): 27-32. doi: 10.3969/j.issn.1673-1689.2018.01.005
[49]	黄燕, 杨玉路, 夏伟等. 重组β-环糊精葡萄糖基转移酶生产偶合糖的工艺优化[J]. 生物工程学报,2021,37(4):1415−1424. [HUANG Y, YANG Y L, XIA W, et al. Optimization of maltooligosyl fructofuranosidesproduction by recombinant β-cyclodextrin glycosyltransferase[J]. Chinese Journal Biotechnology,2021,37(4):1415−1424. HUANG Y, YANG Y L, XIA W, et al. Optimization of maltooligosyl fructofuranosidesproduction by recombinant β-cyclodextrin glycosyltransferase[J]. Chinese Journal Biotechnology, 2021, 37(4): 1415-1424.
[50]	柴宝成, 姜钰琳, 倪晔等. 环糊精葡萄糖基转移酶182位点定点改造催化合成糖基化染料木素[J]. 生物工程学报,2022,38(2):749−759. [CHAI B C, JIANG Y L, NI Y, et al. Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis[J]. Chinese Journal of Biological Engineering,2022,38(2):749−759. CHAI B C, JIANG Y L, NI Y, et al. Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis[J] Chinese Journal of Biological Engineering, 2022, 38 (2): 749-759