利用环糊精糖基转移酶转化合成AA-2G的研究
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摘要
利用环糊精糖基转移酶(CGT-SL)对转化合成2-O-α-D-吡喃葡萄糖基-L-抗坏血酸(AA-2G)的条件进行考察。反应产物通过HPLC/LC-MS分析确定含有AA-2G。以便宜易得的可溶性淀粉和VC作为底物,在初始反应条件下AA-2G的产量为1.23 g/L。通过转化条件优化初步确定了转化反应的最优条件:最适温度15℃,最适pH 4.0,转化反应时间32 h,底物VC和可溶性淀粉的比例为2∶4,VC质量浓度为16 g/L,酶浓度为79 U/m L时,AA-2G的产量达到6.23 g/L,VC转化率为19.93%。
The enzymatic synthesis condition of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid(AA-2G) was studied in this dissertation.AA-2G was synthesized by using cyclodextrin glycosyltransferase(CGT-SL,support by Amano),and soluble starch and VC as the substrate.Under the initial condition,AA-2G production was 1.23 g/L.The best conditions for the synthesis of AA-2G:time 32 h,temperature 15 ℃,pH 4.0,concentration of VC 16 g/L,the proportion of VC and soluble starch is 2 ∶4,the concentration of CGT-SL 79 U/m L.Under these conditions,the final production of AA-2G was 6.23 g/L,and the transformation rate of VC reached 19.93%.
The enzymatic synthesis condition of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid(AA-2G) was studied in this dissertation.AA-2G was synthesized by using cyclodextrin glycosyltransferase(CGT-SL,support by Amano),and soluble starch and VC as the substrate.Under the initial condition,AA-2G production was 1.23 g/L.The best conditions for the synthesis of AA-2G:time 32 h,temperature 15 ℃,pH 4.0,concentration of VC 16 g/L,the proportion of VC and soluble starch is 2 ∶4,the concentration of CGT-SL 79 U/m L.Under these conditions,the final production of AA-2G was 6.23 g/L,and the transformation rate of VC reached 19.93%.
引文
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[15]AGA H,YONEYAMA M,SAKAI S,et al.Synthesis of 2-O-α-D-glucopyranosyl L-ascorbic acid by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus[J].Agricultural and Biological Chemistry,1991,55(7):1751-1756.
[16]韩瑞枝.Paenibacillus macerans环糊精葡萄糖基转移酶的分子改造及其合成糖基化维生素C的研究[D].无锡:江南大学,2013.
[17]LI Z,LI B,GU Z,et al.Extracellular expression and biochemical characterization ofα-cyclodextrin glycosyltransferase from Paenibacillus macerans[J].Carbohydrate Research,2010,345(7):886-892.
[18]李兆丰.软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D].无锡:江南大学,2009.
[19]RUIZHI H.Molecular engineering of cyclodextrin glycosyltransferase and its application on glycoysl Vitamin C synthesis[D].Wu Xi:jiangnan university,2013.
[20]张子臣.酶法转化合成2-氧-α-D-吡喃葡萄糖基抗坏血酸[D].无锡:江南大学,2010.
[2]YAMAMOTO I,TAI A.The current state on development of novel vitamin derivatives[J].Nihon rinsho.Japanese Journal of Clinical Medicine,1999,57(10):2332-2338.
[3]KOUKI M,NORIO M,KYOKO F,et al.Comparison of ascorbic acid and ascorbic acid 2-O-α-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts[J].Biochemical Pharmacology,1992,44(11):2191-2197.
[4]YAMAMOTO I,SUGA S,MITOH Y,et al.Antiscorbutic activity of L-ascorbic acid 2-glucoside and its availability as a vitamin C supplement in normal rats and guinea pigs[J].Journal of Pharmacobio-Dynamics,1990,13(11):688-695.
[5]TATEMOTO H,OOTAKI K,SHIGETA K,et al.Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-α-glucoside during in vitro maturation[J].Biology of Reproduction,2001,65(6):1800-1806.
[6]WAKAMIYA H,SUZUKI E,YAMAMOTO I,et al.In situ intestinal absorption of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid in guinea pigs[J].Journal of Nutritional Science and Vitaminology,1995,41(2):265-272.
[7]YAMASAKI H,NISHI K,MIYAKE T.Process for producing high 2-O-α-D-glucopyranosyl-L-ascorbic acid.US,6576446[P].2003-05-10.
[8]YAMAMOTO I,TANAKA M,MUTO N.Enhancement of in vitro antibody production of murine splenocytes by ascorbic acid2-O-α-glucoside[J].International Journal of Immunopharmacology,1993,15(3):319-325.
[9]ZHENG M,ENDO T,ZIMMERMANN W.Synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases from bacterial isolates[J].Journal of Inclusion Phenomena and Macrocyclic Chemistry,2002,44(1-4):387-390.
[10]TANAKA M,MUTO N,YAMAMOTO I.Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-α-glucoside formation[J].Biochim Biophy Acta,1991,1078(2):127-132.
[11]Van der Veen BA,Van Alebeek GJ,Uitdehaag JC,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,2000,267(3):658-665.
[12]MANDAI T,YONEYAMA M,SAKAI S,et al.The crystal structure and physicochemical properties of L-ascorbic acid2-glucoside[J].Carbohydrate Research,1992,232(2):197-205.
[13]JUN H K,BAE K M,KIM S 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.
[14]ZHANG Z C,LI J H,LIU L,et al.Enzymatic transformation of 2-O-α-D-glucopyranosyl-L-ascorbic acid byα-cyclodextrin glucanotransferase from recombinant Escherichia coli[J].Biotechnology and Bioprocess Engineering,2011,16(1):107-113.
[15]AGA H,YONEYAMA M,SAKAI S,et al.Synthesis of 2-O-α-D-glucopyranosyl L-ascorbic acid by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus[J].Agricultural and Biological Chemistry,1991,55(7):1751-1756.
[16]韩瑞枝.Paenibacillus macerans环糊精葡萄糖基转移酶的分子改造及其合成糖基化维生素C的研究[D].无锡:江南大学,2013.
[17]LI Z,LI B,GU Z,et al.Extracellular expression and biochemical characterization ofα-cyclodextrin glycosyltransferase from Paenibacillus macerans[J].Carbohydrate Research,2010,345(7):886-892.
[18]李兆丰.软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D].无锡:江南大学,2009.
[19]RUIZHI H.Molecular engineering of cyclodextrin glycosyltransferase and its application on glycoysl Vitamin C synthesis[D].Wu Xi:jiangnan university,2013.
[20]张子臣.酶法转化合成2-氧-α-D-吡喃葡萄糖基抗坏血酸[D].无锡:江南大学,2010.