特异的柴胡皂苷糖苷酶的分离纯化

Purification and Characterization of New Special Saikosaponin-glycosidase

研究了微生物Aspergillus oryzaec42菌株产特异的柴胡皂苷糖苷酶的分离纯化及其酶学性质.结果表明,该酶能够水解柴胡皂苷A或柴胡皂苷B2侧链的3-O-β-(1→3)-Glc和3-O-β-Fuc,其分子量约为58000.柴胡皂苷糖苷酶首先水解柴胡皂苷A或B2侧链的3-O-β-(1→3)-Glc生成3-O-β-Fuc-柴胡皂苷元A或B2,然后进一步水解3-O-β-Fuc-柴胡皂苷元A或B2的3-O-β-Fuc生成柴胡皂苷元A或B2.柴胡皂苷糖苷酶的最适反应温度为4℃,最适pH值为5.0;Na+和K+对酶活力的影响不明显;Cu2+,Hg2+和Ag+对酶活力有显著的抑制作用;Ca2+和Mg2+对酶活力有轻微的激活作用.

Radix bupleuri,a well-known traditional Chinese herb-drug,is often used to treat common cold with fever.One of the major physiologically active ingredients in radix bupleuri is saikosaponin which has the properties of anti-inflammation,anti-hypersusceptibility,anti-thrombus.The natural structures of saikosaponins are not suitable for physiological or pharmacological effects,the enzymatic transforming generally improves the bioactivity of natural productions.In this paper,a new special saikosaponin-glycosidase hydrolyzing 3-O-β-（1→3）-glucoside and 3-O-β-fucoside of saikosaponin A or B2 was isolated from Aspergillus oryzae c42 strain,purified and characterized.The methods of centrifugation,ammonium sulfate precipitation,ion-exchange chromatography on DEAE-cellulose column and vertical slab polyacrylamide gel electrophoresis（PAGE） were used to purify the enzyme,and its partial characteristics were studied.The molecular weight of saikosaponin-glycosidase was about 58000 by SDS-PAGE.The enzyme hydrolyzed 3-O-β-（1→3）-glucoside of saikosaponin A or B2 into 3-O-β-fucoside-saikosapogenin A or B2,and further hydrolyzed 3-O-β-fucoside of 3-O-β-fucoside-saikosapogenin A or B2 into their aglycone（saikosapogenin A or B2） exhibiting significant differences from all previously reported glycosidases.The optimum temperature of the saikosaponin-glycosidase was 40 ℃;the optimum pH was 5.0.The activity of this enzyme was not apparently affected by Na＋ and K＋ ions,but it was significantly inhibited by Cu2＋,Hg2＋ and Ag＋ ions;and slightly affected by Ca2＋ and Mg2＋ ions.Further study on saikosaponin-glycosidase will bring new insight to the understanding of glycosidases.