茶叶β-葡萄糖苷酶亲和层析纯化与性质研究

Purification by Affinity Chromatography and Characterization of Tea Leaf β-Glucosidase

以化学合成的β-葡萄糖苷酶的抑制剂β-葡萄糖脒为配体,通过N-糖苷键交联到支持物上,使糖脒N-糖苷键一侧的氨基保持带正电荷的活化状态,合成了一种β-葡萄糖苷酶的亲和层析树脂。这种合成的亲和层析树脂在弱酸条件下非常稳定,能选择性地吸附β-葡萄糖苷酶,并能多次重复使用。利用这种亲和层析树脂,在pH6和pH5条件下,从薮北种茶树叶片中纯化出两种分子量分别为63和75kDa的β-葡萄糖苷酶单体酶。以对硝基苯酚β-葡萄糖苷为底物,研究了两种酶的性质。TLC分析结果表明,这两种β-葡萄糖苷酶都能水解(Z)-3-己烯醇、苯甲醇和苯乙腈的β-D-葡萄糖苷。

Two kinds of β-Glucosidases from Camellia sinensis var. sinensis cv. Yabukita were purified by a sophisticate application of affinity chromatography. An affinity adsorbent for β-Glucosidases has been prepared with a positively charged β-glucosylamidine as a ligand in which a glycon moiety is connected via a N-glycoside linkage. The synthesized affinity adsorbent is quite stable under mildly acidic conditions (pH5~6) and can be used up to 20 times over three months without significant affect on the column performance. Two tea leaf β -Glucosidases have been purified to be monomers with molecular mass of 63 and 75 kDa by affinity chromatography at pH6.0 and pH5.0, respectively. The extensive characterization of tea leaf β-Glucosidasse has been reported. Tea aroma precursors such as benzyl, (Z)-3-hexenyl and D-mandelonitrile β-D-glucopyranosides were hydrolyzed by the purified two β-Glucosidases to liberate tea aroma.