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| 痤疮丙酸杆菌亚油酸异构酶在毕赤酵母中的异源表达 及其静息细胞催化合成共轭亚油酸 |
| Heterologous expression of linoleate isomerase from Propionibacterium acnes in |
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| DOI： |
| 中文关键词: 毕赤酵母 亚油酸异构酶 异源表达 共轭亚油酸 静息细胞催化 |
| 英文关键词:Pichia pastoris linoleate isomerase heterologous expression conjugated linoleic acid resting cell catalysis |
| 基金项目:国家自然科学基金优秀青年基金（31722041） |
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| 中文摘要: |
| 来源于痤疮丙酸杆菌（Propionibacterium acnes）的亚油酸异构酶（PAI）是一种能够通过单酶催化，生成共轭亚油酸（t10,c12-CLA）的亚油酸异构酶。为实现PAI的高效表达，以pPink为表达载体，毕赤酵母（PichiaPinkTM Strain2）为宿主菌，构建毕赤酵母重组菌株（Pichia-pPink-His-opai），实现了PAI在毕赤酵母中的异源表达。对毕赤酵母重组菌进行初步筛选获得蛋白表达量最高的转化子，通过单因素实验优化毕赤酵母重组菌的诱导条件。毕赤酵母重组菌最佳诱导条件为：诱导时间24 h，诱导剂甲醇体积分数2%。在最佳诱导条件下，将毕赤酵母重组菌制成静息细胞催化剂催化合成t10，c12-CLA。在28 ℃、200 r/min、亚油酸质量浓度4 g/L、反应时间6 h条件下，t10,c12-CLA产量为2.12 g/L，转化率可达53%。 |
| 英文摘要: |
| Linoleate acid isomerase (PAI) from Propionibacterium acnes was a linoleate acid isomerase that could generate conjugated linoleic acid (t10, c12-CLA) through single enzyme catalysis. In order to realize the efficient expression of PAI, pPink was used as the expression vector and Pichia pastoris (PichiaPinkTM Strain2) was used as the host strain to construct a Pichia pastoris recombinant strain (Pichia-pPink-His-opai). Finally, the heterologous expression of PAI in Pichia pastoris was realized. The Pichia pastoris recombinant strain was initially screened to obtain the transformant with the highest protein expression. The single factor experiment was used to obtain the best induction conditions for the Pichia pastoris recombinant strain as follows: volume fraction of the inducer methanol 2%, and induction time 24 h. Under these induction conditions, the Pichia pastoris recombinant strain was made into a resting cell catalyst to catalyze the synthsis of t10, c12-CLA. Under the conditions of 28 ℃, 200 r/min, mass concentration of linoleic acid 4 g/L, and reaction time 6 h, the productivity of t10, c12-CLA was 2.12 g/L, and the conversion rate reached 53%. |
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