• 中国中文核心期刊
  • 中国科学引文数据库核心期刊
  • 中国科技核心期刊
  • 中国高校百佳科技期刊
高级检索

罗汉果苷ⅢE的酶法合成

陈玲, 陈依军, 王淑珍, 吴旭日

陈玲, 陈依军, 王淑珍, 吴旭日. 罗汉果苷ⅢE的酶法合成[J]. 中国药科大学学报, 2018, 49(3): 354-359. DOI: 10.11665/j.issn.1000-5048.20180315
引用本文: 陈玲, 陈依军, 王淑珍, 吴旭日. 罗汉果苷ⅢE的酶法合成[J]. 中国药科大学学报, 2018, 49(3): 354-359. DOI: 10.11665/j.issn.1000-5048.20180315
CHEN Ling, CHEN Yijun, WANG Shuzhen, WU Xuri. Enzymatic synthesis of mogroside IIIE[J]. Journal of China Pharmaceutical University, 2018, 49(3): 354-359. DOI: 10.11665/j.issn.1000-5048.20180315
Citation: CHEN Ling, CHEN Yijun, WANG Shuzhen, WU Xuri. Enzymatic synthesis of mogroside IIIE[J]. Journal of China Pharmaceutical University, 2018, 49(3): 354-359. DOI: 10.11665/j.issn.1000-5048.20180315

罗汉果苷ⅢE的酶法合成

基金项目: 国家自然科学基金资助项目(No.81502961);江苏省“六大人才高峰”资助项目(No.SWYY-097)

Enzymatic synthesis of mogroside IIIE

  • 摘要: 为开发罗汉果苷ⅢE的简易制备方法,为开发罗汉果苷类甜味剂提供参考依据。本研究以罗汉果苷Ⅴ为探针筛选糖苷水解酶库,获得了能够区域选择性合成罗汉果苷ⅢE的CPU-GH17,通过考察异丙基硫代半乳糖苷(IPTG)浓度、诱导温度和时间等参数建立了CPU-GH17的高效可溶性表达条件:0.4 mmol/L IPTG、15 ℃和12 h。在此基础上,针对反应pH、温度、酶量、底物浓度以及反应时间等参数进行系统优化,最终确定pH 6.0、45 ℃、3 U/mL酶、5 mg/mL底物和20 h时,罗汉果苷Ⅴ可以完全转化成罗汉果苷ⅢE。本研究成功地开发了基于CPU-GH17的罗汉果苷ⅢE的酶法合成新工艺,并验证了其规模化生产的可行性。
    Abstract: The aim was to develop the simple preparation method of mogroside IIIE, and to lay the foundation for the development of the mogroside sweeteners. In the present study, the glycosidase CPU-GH17, which can regio-selectively biosynthesize mogroside IIIE from mogroside V, was screened from the established library of glycosidases. Then, the soluble expression condition of CPU-GH17 in E. coli was exploited by investigating isopropyl β-D-thiogalactoside(IPTG)concentration, culture temperature and induction time, and 0. 4 mmol/L IPTG, 15 °C and 12 h was used as optimal condition. The result showed that mogroside V could be completely converted into mogroside IIIE under the conditions of pH 6. 0, 45 °C, 3 U/mL enzyme loading, 5 mg/mL substrate concentration for 20 h. In conclusion, a biosynthetic system for the regio-selective preparation of mogroside IIIE by recombinant CPU-GH17 was successfully established and verified at a preparative scale.
  • [1] Wang L,Yang Z,Lu F,et al.Cucurbitane glycosides derived from mogroside IIE:structure-taste relationships,antioxidant activity,and acute toxicity[J].Molecules,2014,19(8):12676-12689.
    [2] Akihisa T,Hayakawa Y,Tokuda H,et al.Cucurbitane glycosides from the fruits of Siraitia grosvenorii and their inhibitory effects on Epstein-Barr virus activation[J].J Nat Prod,2007,70(5):783-788.
    [3] Takemoto T,Arihara S,Nakajima T,et al.Studies on the constituents of fructus Momordicae.I.on the sweet principle[J].Yakugaku Zasshi,1983,103(11):1151-1154.
    [4] Jia Z,Yang X.A minor,sweet cucurbitane glycoside from Siraitia grosvenorii[J].Nat Prod Commun,2009,4(6):769-772.
    [5] Li D,Ikeda T,Huang Y,et al.Seasonal variation of mogrosides in Lo Han Kuo(Siraitia grosvenorii)fruits[J].J Nat Med,2007,61(3):307-312.
    [6] Zhang M,Yang H,Zhang H,et al.Development of a process for separation of mogroside V from Siraitia grosvenorii by macroporous resins[J].Molecules,2011,16(9):7288-7301.
    [7] Itkin M,Davidovich-Rikanati R,Cohen S,et al.The biosynthetic pathway of the nonsugar,high-intensity sweetener mogroside V from Siraitia grosvenorii[J].P Natl Acad Sci 2016,113(47):E7619-E7628.
    [8] Qi T,Ma X,Mo C,et al.An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis[J].Bmc Genomics,2011,12(1):343-355.
    [9] Van LJ, Faijes M, Nieto J, et al. Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus[J].Archaea,2004,1(4):285-292.
    [10] Michlmayr H,Varga E,Malachova A,et al.A versatile family 3 glycoside hydrolase from bifidobacterium adolescentis hydrolyzes β-glucosides of the Fusarium mycotoxins deoxynivalenol,nivalenol,and HT-2 toxin in cereal matrices[J].Appl Environ Microb,2015,81(15):4885-4893.
    [11] Wang H,Yan Y,Lin L,et al.Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides[J].Micro Cell Fact,2016,15(1):134.
    [12] Francis DM,Page R.Strategies to optimize protein expression in E.coli[J].Protoc Protein Sci,2010,5(24):1-29.
    [13] Kaur J,Kumar A,Kaur J.Strategies for optimization of heterologous protein expression in E.coli:roadblocks and reinforcements[J].Int J Biol Macromol,2018,106:803-822.
    [14] Vera C,Guerrero C,Wilson L,et al.Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside[J].Process Biochem,2017,58:128-136.
    [15] Prajapati VS,Trivedi UB,Patel KC.Optimization of glucoamylase production by Colletotrichum sp.KCP1 using statistical methodology[J].Food Sci Biotechnol,2013,22(1):31-38.
    [16] Mooradian AD,Smith M,Tokuda M.The role of artificial and natural sweeteners in reducing the consumption of table sugar:a narrative review[J].Clin Nutr Espen,2017,18:1-8.
计量
  • 文章访问数:  809
  • HTML全文浏览量:  0
  • PDF下载量:  1030
  • 被引次数: 0
出版历程
  • 刊出日期:  2018-06-24

目录

    /

    返回文章
    返回
    x 关闭 永久关闭