两种耐热磷酸化酶的构建及高效催化合成红景天苷
|
摘要
目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。
Objective: Thermostable cellobiose phosphorylase and sucrose phosphorylase were produced by recombinant strain E.coli BL21/pET-Spase and E.coli BL21/pET-Cpase. By combined use of these two phosphorylases, salidroside was efficiently synthesized from inexpensive sucrose. Methods: First, thermostable cellobiose phosphorylase and sucrose phosphorylase recombinant strains were constructed. Then two phosphorylases with sucrose, tyrosol and phosphoric acid were combined. Finally, the mixture was reacted and produced salidroside. Results: At optimal conditions, 23.7 g/L of salidroside was formed over 32 h from 1200 U/L thermostable sucrose phosphorylase, 500 U/L thermostable cellobiose phosphorylase, 110 g/L sucrose, 30 g/L Tyrosol and 50 m M phosphoric acid at the pH7.0 and 45 ℃. Conclusion: In this study, thermostable cellobiose phosphorylase and sucrose phosphorylase were employed as the catalyst, and salidroside was successfully synthesized in high yield. And the thermostable phosphorylases constructed in this study had high activity, which offers a new way to expand the ability of phosphorylases in preparation of β-D-glucosides.
Objective: Thermostable cellobiose phosphorylase and sucrose phosphorylase were produced by recombinant strain E.coli BL21/pET-Spase and E.coli BL21/pET-Cpase. By combined use of these two phosphorylases, salidroside was efficiently synthesized from inexpensive sucrose. Methods: First, thermostable cellobiose phosphorylase and sucrose phosphorylase recombinant strains were constructed. Then two phosphorylases with sucrose, tyrosol and phosphoric acid were combined. Finally, the mixture was reacted and produced salidroside. Results: At optimal conditions, 23.7 g/L of salidroside was formed over 32 h from 1200 U/L thermostable sucrose phosphorylase, 500 U/L thermostable cellobiose phosphorylase, 110 g/L sucrose, 30 g/L Tyrosol and 50 m M phosphoric acid at the pH7.0 and 45 ℃. Conclusion: In this study, thermostable cellobiose phosphorylase and sucrose phosphorylase were employed as the catalyst, and salidroside was successfully synthesized in high yield. And the thermostable phosphorylases constructed in this study had high activity, which offers a new way to expand the ability of phosphorylases in preparation of β-D-glucosides.
引文
[1]王晓晨,魏清筠,李文赟,等.化学全合成红景天苷的研究进展[J].中国抗生素杂志,2018,43(7):786-793
[2]Linh P T,Kim Y H,Hong S P,et al.Quantitative determination of salidroside and tyrosol from the underground part of rhodiolarosea by high performance liquid chromatography[J].Arch.Pharm.Res.,2000,23(4):349-352
[3]蒲忠慧,陈希文,王雄清,等.均匀设计法优选红景天苷提取工艺的研究[J].食品研究与开发,2016,37(14):51-54
[4]纪明江,李玉锋,杨文韬,等.红景天苷提取工艺的研究[J].药物生物技术,2014,21(6):554-557
[5]Zhang H,Pereira B,Li Z,et al.Engineering Escherichia coli coculture systems for the production of biochemical products[J].PNAS,USA,2015,112(27):8266-8271
[6]王梦亮,姜骉麟,崔晋龙,等.表面活性剂在水-有机两相体系中对β-葡萄糖苷酶催化合成红景天苷的影响[J].分子催化,2013,27(1):83-88
[7]何丽妃,薛原楷,石贤爱,等.特殊反应体系中交联β-葡萄糖苷酶聚集体催化合成红景天苷[J].过程工程学报,2015,15(2):313-317
[8]Shi L,Wang C,Zhou X,et al.Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata[J].Plant Cell,Tiss.Org.(PCTOC),2013,114(3):295-303
[9]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
[10]韩雪娇,郭娜,朱美宣,等.红景天苷药理作用及其作用机理研究进展[J].中国生化药物杂志,2015,35(1):171-176
[11]张明发,沈雅琴.红景天苷的神经保护作用及作用机制研究进展[J].药物评价研究,2017,40(7):1019-1028
[12]张伟,赵俊杰,李涛,等.红景天苷对神经退行性疾病作用的研究进展[J].湖南中医药大学学报,2016,36(7):86-89
[13]魏胜华,钱伟,周清华,等.β-葡萄糖苷酶纳米凝胶非水相合成红景天苷[J].化工进展,2018,37(2):694-701
[14]Yu H L,Xu J H,Lu W Y,et al.Environmentally benign synthesis of natural glycosides using apple seed meal as green and robust biocatalyst[J].J.Biotechnol.,2008,133(4):469-477
[15]Hiroyuki A E,Kawahara M K,et al.Synthesis of naturally occurringβ-D-glucopyranoside based on enzymaticβglycosidation[J].J.Mol.Catal.B:Enzym.,2006,40(1-2):8-15
[16]Zhang C,Yu H,Lu M,et al.Enzymic synthesis of salidroside:purification and characterization of salidrosidase from Aspergillas niger[J].Process Biochem.,2005,40(9):3143-3147
[17]Winter K.D,Renterghem L V,Wuyts K,et al.Chemoenzymatic Synthesis ofβ-d-Glucosides using Cellobiose Phosphorylase from Clostridium thermocellum[J].Adv.Synth.Catal.,2015,357:1961-1 969
[18]Wildberger P,Aish G A,Jakeman.D L,et al.Interplay of catalytic subsite residues in the positioning ofα-d-glucose 1-phosphate in sucrose phosphorylase[J].Biochem and Biophy Rep.,2015,2:36-44
[19]Valikhani D,Bolivar J M,Pfeiffer M,et al.Multivalency Effects on the Immobilization of Sucrose Phosphorylase in Flow Microchannels and Their Use in the Development of a High-Performance Biocatalytic Microreactor[J].ChemCatChem,2016,9(1):161-166
[20]Kwon T,Kim C T,Lee J H.Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase[J].Biotechnol.Lett.,2007,29:611-615
[21]Gudiminchi R K,Nidetzky B.Walking a Fine Line with Sucrose Phosphorylase:Efficient Single-Step Biocatalytic Production of L-Ascorbic Acid 2-Glucoside from Sucrose[J].Chem Bio Chem,2017,18(14):1387-1390
[22]Kitao S,Sekine H.α-D-Glucosyl Transfer to Phenolic Compounds by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production ofα-Arbutin[J].Biosci.Biotechnol.Biochem.,1994,58(1):38-42
[23]李群良,张欣英,杨克迪,等.巨大芽孢杆菌Bacillus megaterium NCIB8508蔗糖磷酸化酶生物催化合成α-熊果苷的研究[J].化学与生物工程,2011,28(4):46-51
[24]Bolivar J M,Luley-Goedl C,Leitner E,et al.Production of glucosyl glycerol by immobilized sucrose phosphorylase:Options for enzyme fixation on a solid support and application in microscale flow format[J].J.Biotechnol,2017,257(9):131-138
[25]Hiroyuki N,Maher A H,Bent O P,et al.Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum[J].Biochimie,2010,92:1818-1826
[26]Tom V,Dirk A,Margo D,et al.The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity[J].Appl.Microbiol.Biotechnol.,2014,98(16):7027-7037
[2]Linh P T,Kim Y H,Hong S P,et al.Quantitative determination of salidroside and tyrosol from the underground part of rhodiolarosea by high performance liquid chromatography[J].Arch.Pharm.Res.,2000,23(4):349-352
[3]蒲忠慧,陈希文,王雄清,等.均匀设计法优选红景天苷提取工艺的研究[J].食品研究与开发,2016,37(14):51-54
[4]纪明江,李玉锋,杨文韬,等.红景天苷提取工艺的研究[J].药物生物技术,2014,21(6):554-557
[5]Zhang H,Pereira B,Li Z,et al.Engineering Escherichia coli coculture systems for the production of biochemical products[J].PNAS,USA,2015,112(27):8266-8271
[6]王梦亮,姜骉麟,崔晋龙,等.表面活性剂在水-有机两相体系中对β-葡萄糖苷酶催化合成红景天苷的影响[J].分子催化,2013,27(1):83-88
[7]何丽妃,薛原楷,石贤爱,等.特殊反应体系中交联β-葡萄糖苷酶聚集体催化合成红景天苷[J].过程工程学报,2015,15(2):313-317
[8]Shi L,Wang C,Zhou X,et al.Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata[J].Plant Cell,Tiss.Org.(PCTOC),2013,114(3):295-303
[9]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
[10]韩雪娇,郭娜,朱美宣,等.红景天苷药理作用及其作用机理研究进展[J].中国生化药物杂志,2015,35(1):171-176
[11]张明发,沈雅琴.红景天苷的神经保护作用及作用机制研究进展[J].药物评价研究,2017,40(7):1019-1028
[12]张伟,赵俊杰,李涛,等.红景天苷对神经退行性疾病作用的研究进展[J].湖南中医药大学学报,2016,36(7):86-89
[13]魏胜华,钱伟,周清华,等.β-葡萄糖苷酶纳米凝胶非水相合成红景天苷[J].化工进展,2018,37(2):694-701
[14]Yu H L,Xu J H,Lu W Y,et al.Environmentally benign synthesis of natural glycosides using apple seed meal as green and robust biocatalyst[J].J.Biotechnol.,2008,133(4):469-477
[15]Hiroyuki A E,Kawahara M K,et al.Synthesis of naturally occurringβ-D-glucopyranoside based on enzymaticβglycosidation[J].J.Mol.Catal.B:Enzym.,2006,40(1-2):8-15
[16]Zhang C,Yu H,Lu M,et al.Enzymic synthesis of salidroside:purification and characterization of salidrosidase from Aspergillas niger[J].Process Biochem.,2005,40(9):3143-3147
[17]Winter K.D,Renterghem L V,Wuyts K,et al.Chemoenzymatic Synthesis ofβ-d-Glucosides using Cellobiose Phosphorylase from Clostridium thermocellum[J].Adv.Synth.Catal.,2015,357:1961-1 969
[18]Wildberger P,Aish G A,Jakeman.D L,et al.Interplay of catalytic subsite residues in the positioning ofα-d-glucose 1-phosphate in sucrose phosphorylase[J].Biochem and Biophy Rep.,2015,2:36-44
[19]Valikhani D,Bolivar J M,Pfeiffer M,et al.Multivalency Effects on the Immobilization of Sucrose Phosphorylase in Flow Microchannels and Their Use in the Development of a High-Performance Biocatalytic Microreactor[J].ChemCatChem,2016,9(1):161-166
[20]Kwon T,Kim C T,Lee J H.Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase[J].Biotechnol.Lett.,2007,29:611-615
[21]Gudiminchi R K,Nidetzky B.Walking a Fine Line with Sucrose Phosphorylase:Efficient Single-Step Biocatalytic Production of L-Ascorbic Acid 2-Glucoside from Sucrose[J].Chem Bio Chem,2017,18(14):1387-1390
[22]Kitao S,Sekine H.α-D-Glucosyl Transfer to Phenolic Compounds by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production ofα-Arbutin[J].Biosci.Biotechnol.Biochem.,1994,58(1):38-42
[23]李群良,张欣英,杨克迪,等.巨大芽孢杆菌Bacillus megaterium NCIB8508蔗糖磷酸化酶生物催化合成α-熊果苷的研究[J].化学与生物工程,2011,28(4):46-51
[24]Bolivar J M,Luley-Goedl C,Leitner E,et al.Production of glucosyl glycerol by immobilized sucrose phosphorylase:Options for enzyme fixation on a solid support and application in microscale flow format[J].J.Biotechnol,2017,257(9):131-138
[25]Hiroyuki N,Maher A H,Bent O P,et al.Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum[J].Biochimie,2010,92:1818-1826
[26]Tom V,Dirk A,Margo D,et al.The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity[J].Appl.Microbiol.Biotechnol.,2014,98(16):7027-7037