韭菜β-木糖苷酶的分离纯化与部分性质研究

Isolation, Purification and Characterization of β-D-Xylosidase from Leek

新鲜韭菜经匀浆、缓冲液提取、硫酸铵分级沉淀、羧甲基离子交换层析(carboxymethyl-sephar ose,CMSepharose)再通过Su perdex-200凝胶过滤层析,从而获得电泳纯的β-木糖苷酶。该酶的比活力达到18.25 U/mg,纯化倍数为12.59,回收率为1.83%,分子质量为123.02 k D,亚基分子质量为61.51 k D。通过对β-木糖苷酶的酶学性质研究得到最适温度为65℃,最适p H值为4。该酶在25~55℃及p H 3.0~5.0的范围内有较好的稳定性;在最适条件下测得其米氏常数(K_m)值为0.28 mmol/L;甲醇、乙醇、异丙醇及十二烷基硫酸钠(sodium dodecyl sulfate,SDS)和Ag+对该酶具有抑制作用,Mn^(2+)和Co^(2+)对该酶具有一定的激活作用。

β-D-xylosidase of electrophoretic purity was obtained from leak after homogenization, buffer extraction, ammonium sulfate fractionat ion precipitation, CM-Sepharose ion-exchange and Superdex-200 gel filtration chromatography. Our results showed that the specific activity of β-D-xylosidase was 18.25 U/mg, purification fold was 12.59, and recovery rate was 1.83% after purification. The relative molecular weight of β-D-xylosidase was approximately 123.02 k D, in which the subunit molecular mass was 61.51 k D. Enzymatic properties of β-D-xylosidase showed that the optimal temperature and p H were 65 ℃ and 4.0, respectively. It was relatively stable in the range of 25–55 ℃ and p H 3.0–5.0, respectively. Furthermore, its K_m was 0.28 mmol/L under the optimum conditions. The activity of β-D-xylosidase could be inhibited by methanol, ethanol, isopropanol and so dium dodecyl sulfate and sodium salt（SDS） as well as Ag＋, and activated by Mn^（2＋） and Co^（2＋）.