| 王淮,罗文浩,普元倩,徐春萍,杨健康.菊粉内切酶在毕赤酵母中的表达及酶学性质测定[J].井冈山大学自然版,2020,41(3):34-38 |
| 菊粉内切酶在毕赤酵母中的表达及酶学性质测定 |
| EXPRESSION OF ENDOINULINASE IN PICHIA PASTORIS AND DETERMINATION OF ITS ENZYMATIC PROPERTIES |
| 投稿时间：2020-01-09 修订日期：2020-03-12 |
| DOI：10.3969/j.issn.1674-8085.2020.03.007 |
| 中文关键词: 毕赤酵母 菊粉内切酶 表达 酶学性质 水解产物 |
| 英文关键词: Pichia pastoris endoinulinase expression enzymatic properties hydrolysate |
| 基金项目:大理大学博士科研启动基金项目（KYBS201603） |
| 作者 | 单位 | | 王淮 | 大理大学基础医学院, 云南, 大理 671000 | | 罗文浩 | 中山大学光华口腔医学院, 广东, 广州 510080 | | 普元倩 | 大理大学基础医学院, 云南, 大理 671000 | | 徐春萍 | 大理大学基础医学院, 云南, 大理 671000 | | 杨健康 | 大理大学基础医学院, 云南, 大理 671000 |
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| 中文摘要: |
| 为了实现菊粉内切酶在毕赤酵母（Pichia pastoris）中的高活性表达并对表达的菊粉内切酶酶学性质、水解菊粉的产物进行分析。通过下载无花果曲霉菊粉内切酶基因的编码序列，做了密码子优化后进行全基因合成，然后在毕赤酵母GS115中表达。对表达的菊粉内切酶的酶活、最适反应温度、最适pH值、热稳定性进行了分析，最后对酶水解菊粉的产物进行了高效液相色谱（HPLC）分析。摇瓶表达的酶活力为420 U/mL，酶的最适反应温度为55℃，最适反应pH为6.0。HPLC分析表明：表达的菊粉内切酶酶解底物菊粉的主要产物为低聚果糖，聚合度为3~5之间。构建的工程酵母可以高效表达菊粉内切酶，可以用于生产低聚果糖。 |
| 英文摘要: |
| To achieve high activity expression of endoinulinase in Pichia pastoris, and to analyze the enzymatic properties and the products of hydrolyzed inulin, we downloaded the coding sequence of the endoinulinase gene of Aspergillus ficuum, performed the whole gene synthesis after codon optimization, and expressed in P. pastoris GS115. The enzymatic activity, optimum reaction temperature, optimum pH value and thermal stability were analyzed. Finally, the products of inulin hydrolysis were analyzed by high performance liquid chromatography (HPLC). The enzymatic activity expressed in shake flask was 420 U/ml. The optimum reaction temperature of the enzyme was 55℃, and the optimum pH was 6.0. HPLC analysis showed that the main products of inulin hydrolysis by endoinulinase were oligofructoses with a degree of polymerization of 2~5. The constructed genetically engineered yeast can efficiently express endoinulinase and be used to produce oligofructose. |
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