摘要
采用酶促转化方法，以维生素C（VC）和糖基供体为转化反应底物，重组E.coli BL21（DE3）-pET30a-cgt1为产酶菌株，对产酶条件和转化合成2-O-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G）的条件进行考察。研究结果表明∶反应产物通过HPLC分析确定含有AA-2G。诱导温度为25 ℃，发酵培养基为TB液体培养基，诱导时长为8 h，接种量为4 %，IPTG诱导浓度为50 μM，诱导时菌体密度为1.2-1.9，培养基初始pH值为自然pH值时，重组菌株酶表达量最高且终菌体密度最大。以β-环糊精和VC作为反应底物，在初始反应条件下AA-2G的产量为2.21 g/L，VC转化率为23.02%。通过对其转化反应条件进行优化，初步确定了转化反应的最佳条件∶转化反应pH值为5.0，转化反应时间为48 h，转化反应温度为30 ℃，转化反应溶剂为纯水，粗酶液添加量与转化反应总体积比v（粗酶液）∶v（转化反应总体积）=1∶5，VC与β-环糊精的质量比m（VC）∶m（β-环糊精）=1∶2，葡萄糖淀粉酶添加量为50U/mL，葡萄糖淀粉酶水解反应时间为16h时，AA-2G的产量达到68.61g/L，VC转化率为71.45%。
关键词： α-环糊精葡萄糖基转移酶（α-CGTase）；2-O-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G）；酶促合成
Abstract
The enzyme-induced transformation method was used, with vitamin C (VC) and glucose donor as the substrate, and recombinant E.coli BL21(DE3)-pET30a-cgt1 as the enzyme-producing strain. The enzyme-producing conditions and the synthesis conditions of 2-O-α-D-glucopylanose-L-ascorbic acid (AA-2G) were investigated. The results showed that the reaction product was determined to contain AA-2G by HPLC analysis. The induction temperature was 25 ℃, the fermentation medium was TB liquid medium, the induction time was 8 h, the inoculation amount was 4 %, the IPTG induction concentration was 50 μM, the cell density of the recombinant strain was 1.2-1.9, the initial pH value of the medium was natural pH value, the enzyme expression of the recombinant strain was the highest and the terminal bacterial cell density was the highest. With β-cyclodextrin and VC as substrates, the yield of AA-2G was 2.21 g/L and the conversion of VC was 23.02 % under the initial reaction conditions. By optimizing the transformation reaction conditions, the optimum conditions for the transformation reaction were preliminarily determined: The pH value of the conversion reaction was 5.0, the conversion reaction time was 48 h, the conversion reaction temperature was 30 ℃, the conversion reaction solvent was pure water, v(crude enzyme solution):v(total transformation reaction volume) = 1:5, m(VC):m(β-cyclodextrin)=1:2, and the addition amount of glucose amylase was 50 U/mL. When the hydrolysis time of glucose amylase was 16 h, the yield of AA-2G reached 68.61 g/L and the conversion rate of VC was 71.45 %.
Key words： α-cyclodextrin glucosyltransferase(α-CGTase); 2-O-alpha-D-glucopyranosyl-L-ascorbic acid(AA-2G); Enzymatic synthesis
参考文献 References
[1] 张子臣. 酶法转化合成2-氧-α-D-吡喃葡萄糖基抗坏血酸. 无锡:江南大学,2010.
[2] H Tatemoto, K Ootaki, et al. Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation. Biol Reprod, 2001, 65(6):1800-6.
[3] I Yamamoto, N Muto, et al. Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid. J Nutr, 1992, 122(4):871-7.
[4] Zhankun Wang, Qingsheng Qi, et al. Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production. Appl Environ Microbiol, 2006 ,72(3):1873-7.
[5] 邢琳,张秀华,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. 中国生化药物杂志,2016,36(11):5-8.
[6] 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．无锡: 江南大学 2013
[7] 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. 食品与生物技术学报,2018,37(01): 27-32.
[8] Kai Song, Jingjing Sun, et al. Heterologous expression of cyclodextrin glycosyltransferase my20 in Escherichia coli and Its application in 2-O-α-D-glucopyranosyl-L-ascorbic Acid production. Front Microbiol, 2021, 12:664339.
[9] Xiumei Tao, Tian Wang, et al. Enhanced 2 O α D Glucopyranosyl L ascorbic Acid Synthesis through Iterative Saturation Mutagenesis of Acceptor Subsite Residues in Bacillus stearothermophilus NO2 Cyclodextrin Glycosyltransferase. J Agric Food Chem, 2018, 66(34):9052-9060.