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| 理性设计提高华根霉脂肪酶对不饱和长链脂肪酸的底物特异性及其在大豆油水解中的应用 |
| Enhancement of substrate specificity towards unsaturated long-chain fatty acids of Rhizopus chinensis lipase through rational efficient hydrolysis of soybean oil design for |
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| DOI： |
| 中文关键词: 脂肪酶 脂肪酸 理性设计 底物特异性 大豆油水解 |
| 英文关键词:lipase fatty acid rational design substrate specificity hydrolysis of soybean oil |
| 基金项目: |
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| 中文摘要: |
| 为了提高华根霉脂肪酶在大豆油水解制备脂肪酸中的应用效果，通过理性设计的方法改造脂肪酶的脂肪酸特异性。对突变酶的酶学性质进行了研究，并通过单因素实验优化脂肪酶催化水解大豆油的反应条件。结果表明：利用定点突变技术获得了一系列脂肪酸特异性改变的脂肪酶，其中突变酶HQL增强了对不饱和长链脂肪酸的特异性，并且对p-NPP(C16)的水解活力相比野生型提高了2.72倍；所有突变酶的最适温度和最适pH均为40?℃和8.0，与野生型脂肪酶一致；得到脂肪酶催化水解大豆油的最适反应条件为反应时间24 h、水油质量比1∶?1、加酶量500 U/g（以油质量计）、pH 8.0、温度40?℃，在最适反应条件下，野生型酶催化大豆油水解率仅为80%，而突变酶HQL由于增强了对长链不饱和脂肪酸的底物特异性，对大豆油水解率提高到98%。 |
| 英文摘要: |
| Fatty acid specificity of Rhizopus chinensis lipase was altered by rational design for efficient hydrolysis of soybean oil. The enzymatic properties of these variants were studied. The reaction conditions of hydrolysis of soybean oil by the lipase were optimized by single factor experiment. The results showed that a series of variants were obtained by site-directed mutagenesis. Compared with the wild-type lipase, the variant HQL showed enhanced specificity towards unsaturated fatty acids and increased hydrolytic activity for p-NPP(C16) by 2.72 fold. The optimal temperature and optimal pH of the variants were 40?℃ and 8.0, which were consistent with the wild-type lipase. The optimal reaction conditions of hydrolysis of soybean oil by the lipase were obtained as follows: reaction time 24 h, mass ratio of water to oil 1∶?1, dosage of lipase 500 U/g (based on oil mass),pH 8.0 and temperature 40?℃. Under the optimal reaction conditions, the hydrolysis rate of soybean oil by the variant HQL increased to 98% due to the increasing specificity towards unsaturated long-chain fatty acids, while it was only 80% for the wild-type lipase. |
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