射干中β-葡萄糖苷酶的提取纯化和酶学性质研究  

Extraction.Purification and Enzymology Properties ofβ-glucosidase in Belamcandae Rhizoma

作　　者：辛旭阳 张微微 尤献民[1] 刘晶[1] 邸子真[1] 李国信[1] 邹桂欣[1] 甘雨[1] XIN Xu-yang;ZHANG Wei-wei;YOU Xian-min;LIU Jing;DI Zhi-zhen;LI Guo-xin;ZOU Gui-xin;GAN Yu(Liaoning Provincial Academy of Traditional Chinese Medicine,Shenyang Liaoning,110034,China;Boenringer-lngelheim,American,06877)

机构地区：[1]辽宁省中医药研究院,辽宁沈阳110034 [2]勃林格殷格翰,美国06877

出　　处：《时珍国医国药》2023年第7期1612-1615,共4页Lishizhen Medicine and Materia Medica Research

基　　金：国家“十三五”新药专项项目(2017ZX09301019);辽宁省教育厅项目(L202031);辽宁省沈阳市中青年科技创新人才支持计划(RC210047);辽宁中医药大学科研项目(2021LZY020)。

摘　　要：目的提取纯化射干中β-葡萄糖苷酶粗酶,测定酶活性,验证酶的水解作用;并对粗酶的基本酶学性质进行初步研究。方法采用缓冲盐溶液低温提取射干中β-葡萄糖苷酶粗酶,用透析膜进行纯化;以对硝基苯酚-β-D葡萄糖苷为底物测定β-葡萄糖苷酶活性;验证其对野鸢尾苷及射干药材酶解作用;对其pH值稳定性、热稳定性及放置稳定性等酶学性质进行初步研究。结果透析膜法可以较好的纯化粗酶,以pNPG为底物,在pH值为5.0,温度为50℃时,粗酶活性为25 U·L^(-1);粗酶可以定向将野鸢尾苷对照品及射干药材中野鸢尾苷定向转化为野鸢尾黄素;基本酶学性质初步研究结果表明,该酶在pH值3.0~5.4之间时,相对酶活性均在84%以上;在30~50℃时,其酶活力热稳定性较好;酶的保存稳定性4℃可以保存18 d。结论射干中可能含有β-葡萄糖苷酶,该酶能将射干异黄酮苷定向转化为异黄酮苷元,可为射干酶定向炮制研究提供参考。Objectiveβ-glucosidase crude enzyme was extracted and purified from Belamcandae Rhizoma,its activity was measured,the hydrolysis of the enzyme was verified,and the basic enzymatic properties of crude enzyme were studied.Methodsβ-glucosidase crude enzyme was extracted by buffer salt solution at low temperature and purified by dialysis membrane.β-glucosidase activity was determined by using p-nitrophenol-β-D glucoside as substrate.The enzymatic hydrolysis effects on irisin and Belamcandae Rhizoma were verified.The enzymatic properties such as pH stability,thermal stability and placement stability were studied.Results The dialysis membrane method could purify the crude enzyme well.When pNPG was used as substrate,the crude enzyme activity was 25 U·L^(-1)at pH 5.0 and temperature 50℃.The crude enzyme can directionally convert irisin to Irigenin.The results of preliminary study on basic enzymatic properties showed that the relative activity of the enzyme was above 84%when the pH 3.0~5.4.The thermal stability of the enzyme activity was better at 30~50℃.The storage stability of the enzyme can be maintained for 18 days at 4℃.Conclusion Belamcandae Rhizoma may containβ-glucosidase,which can convert isoflavone glycosides to isoflavone aglycones,The results may provide a reference for the study of the directional processing of isoflavone by enzymes.

关 键 词：射干 Β-葡萄糖苷酶 pNPG 定向转化 

分 类 号：R284.2[医药卫生—中药学]