| 摘要: |
| 采用硫酸铵分级沉淀、凝胶过滤和离子交换层析等方法，对毕赤酵母工程菌发酵液中的原果胶酶进行了分离纯化，测定了其分子质量，并确定了离子交换层析法的最佳离子交换条件。结果表明，硫酸铵的饱和度为70％时，可以使原果胶酶的回收率达到71．9％，比活力为1096．35 U/mg；经Sephadex G75凝胶过滤，原果胶酶回收率达到57．6％，酶的比活力提高到3762．40 U/mg；最佳离子交换条件为：0～0．5 mol/L NaCl（缓冲休系为NoAc—HAc，pH5．8）溶液线性洗脱、Sepharose Fast Flow离子交换层析分离．在该条件下酶的比活力提高到9743．20 U/mg，纯度为粗酶液的18．91倍。达到了电泳级；SDS-PAGE电泳结果表明，其分子质量为43．17 ku。 |
| 关键词: 毕赤酵母 原果胶酶 分离纯化 |
| DOI： |
| 分类号:TS201.2 |
| 基金项目:江苏省高技术项目（BG2002319） |
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| Purification of protopectinase from Pichia pastoris engineering strain |
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LIU Zhan-min LU Zhao-xin L(U) Feng-xia BIE Xiao-mei ZHAO Hai-zhen
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| Abstract: |
| The electrophoresis level protopectinase was purified by ammonium sulfate,Sephadex G75 gel filter and ion exchange chromatography from Pichia pastoris engineering strain. The experiment showed that the recovery of protopectinase could reach 71.9% and specific activity was 1 096.35 U/mg when saturation 70% ammonium sulfate precipitated protopectinase. The recovery of protopectinase was 57.6% and specific activity reached 3 762.40 U/mg by Sephadex G75 gel filter. Finally,with the ion exchange chromatography the protopectinase was purified 18.91 times than the crude protopectinase and its specific activity increased to 9 743.20 U/mg,SDS-PAGE electrophoresis showed its molecular weight was 43.17 ku. |
| Key words: Pichia pastoris protopectinase purification |
