中国中药杂志

2018, v.43(08) 1602-1609

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3种嗜热β-葡萄糖苷酶催化黄芩苷生产黄芩素的研究
Comparison of three different thermophilic beta-glucosidases for baicalin transformation

吴涛;张珊珊;裴建军;萧伟;王振中;赵林果;
WU Tao;ZHANG Shan-shan;PEI Jian-jun;XIAO Wei;WANG Zhen-zhong;ZHAO Lin-guo;Co-innovation Center for Sustainable Forestry in Southern China,Nanjing Forestry University;College of Chemical Engineering,Nanjing Forestry University;Jiangsu Kanion Pharmaceutical Co.,Ltd.;

摘要(Abstract):

该文分析了来自嗜热菌Thermotoga thermarum DSM 5069的Ttebgl3和Thermotoga petrophila RKU~(-1)的Tpebgl1,Tpebgl3等3种β-葡萄糖苷酶基因及其序列,比较了3种β-葡萄糖苷酶催化转化黄芩苷的最适温度、p H以及DMSO、金属离子对催化转化黄芩苷的影响,并比较了3种β-葡萄糖苷酶转化黄芩苷的动力学常数。结果表明,Tpebgl1,Tpebgl3,Ttebgl3转化黄芩苷的最适pH分别为4.5,5.0,5.5,最适温度为85,80,80℃。Tpebgl3酶和Ttebgl3酶催化转化黄芩苷时对DMSO的耐受性较好。金属离子对3种β-葡萄糖苷酶催化转化黄芩苷的激活作用不明显,GH3家族β-葡萄糖苷酶受到的抑制作用明显强于GH1家族。3种β-葡萄糖苷酶催化黄芩苷的动力学常数存在较大差异,Tpebgl1,Tpebgl3,Ttebgl3酶的K_m分别为0.029 2,0.268 6,0.391 8 mmol·L~(-1),V_(max)分别为4.85,121.04,308.90 U·mg~(-1)。Tpebg1,Tpebgl3,Ttebgl3酶分别在其最适pH,温度下转化0.02 g黄芩苷,3 h后转化率分别达到68%,97.3%,97.31%,且GH3家族的酶用量少、催化效率高。研究结果为高效酶的获得及规模化生物制备黄芩素提供了技术支撑。
Three different beta-glycosidase sequences of Ttebgl3,Tpebgl1 and Tpengl3 from Thermotoga thermarum DSM 5069 and Thermotoga petrophila RKU~(-1) were analyzed. Also,the influence of temperature,p H,concentration of DMSO,metal ions and kinetic constant on catalytic conversion of baicalin had been compared. The results indicated that the optimal p H and optimum temperature for transformation of baicalin was 4. 5 85 ℃,5. 0 80 ℃ and 5. 5 80 ℃,respectively. The family GH3 beta-glycosidase Ttebgl3 and Tpebgl3 had the better DMSO tolerance. The activation effect of the metal ions on the catalytic conversion of baicalin was not obvious,and the inhibition of the GH3 family beta glucosidase was significantly stronger than that of the GH1 family. The kinetic constants of three different beta-glucosidases catalyzed baicalin were significantly different. The K_m and V_(max) values of Tpebgl1,Tpebgl3 and Ttebgl3 were0. 029 2 mmol·L~(-1)4. 85 U·mg~(-1),0. 268 6 mmol·L~(-1)121. 04 U·mg~(-1) and 0. 391 8 mmol·L~(-1)308. 90 U·mg~(-1),respectively. Family GH3 beta-glycosidase converted more baicalin than family GH1 with the optimal conditions,0. 02 g baicalin,and the conversion rate was 68%,97. 3%,97. 31% respectively. The results of the study provided a guarantee for the transformation of baicalin.

关键词(KeyWords): 黄芩苷;黄芩素;β-葡萄糖苷酶;催化转化
baicalin;baicalein;beta-glucosidase;catalytic conversion

Abstract:

Keywords:

基金项目(Foundation): 国家重点研发计划项目(2016YFD0600805);; 林业科学技术推广项目([2017]10号)

作者(Author): 吴涛;张珊珊;裴建军;萧伟;王振中;赵林果;
WU Tao;ZHANG Shan-shan;PEI Jian-jun;XIAO Wei;WANG Zhen-zhong;ZHAO Lin-guo;Co-innovation Center for Sustainable Forestry in Southern China,Nanjing Forestry University;College of Chemical Engineering,Nanjing Forestry University;Jiangsu Kanion Pharmaceutical Co.,Ltd.;

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