假坚强芽胞杆菌中乙醇降解相关酶的克隆、表达及酶学特性

Cloning，expression and characterization of alcohol dehydrogenase and aldehyde dehydrogenase from Bacillus pseudofirmus OF4

摘要:

摘要:【目的】研究假坚强芽胞杆菌OF4中乙醇脱氢酶和乙醛脱氢酶的酶学特性。【方法】通过引物设计，采用PCR技术从嗜碱芽胞杆菌OF4的基因组DNA中扩增获得乙醇脱氢酶(adh)基因和乙醛脱氢酶(aldh)基因，构建表达载体，通过异源原核表达，Ni-NTA柱层析纯化酶蛋白，分析其酶学特性。【结果】乙醛脱氢酶的最适反应温度为35℃，最适反应pH值为8.0，酶蛋白的活力为979.6 U/mg，其稳定性在25℃和35℃下比45℃稍好;尽管由于乙醇脱氢酶的表达量低而未能纯化获得酶蛋白，但通过双基因共表达及乙醇耐受性实验发现乙醇脱氢酶也具备较高的催化活性。【结论】成功地从假坚强芽胞杆菌OF4中克隆获得了乙醇脱氢酶和乙醛脱氢酶基因，二者共同作用能够较大提高宿主对乙醇的耐受性。

关键词:关键词:假坚强芽胞杆菌，乙醛脱氢酶，乙醇脱氢酶，乙醇耐受性，共表达

Abstract:

Abstract:［Objective］ To clone and characterize the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from Bacillus pseudofirmus OF4． ［Methods］Genes of adh and aldh were cloned by PCR; expression vectors pET-Ahd and pET-Aldh were constructed and expressed in Escherichia coli BL21 (DE3)．After Ni-NTA column chromatography purification，the protein was characterized．［Results］The optimal temperature and pH of ALDH was 35℃ and 8. 0，the specific activities of ALDH was 979.6 U/mg protein，the thermostability at 25℃ and 35℃ was better than at 45℃ ． Although the expression level of ADH was too low to purify，but it was found that ADH had high catalytic activities by experiments of co-expression and ethanol tolerance．［Conclusion］Adh and aldh from B． pseudofirmus OF4 were cloned successfully． Co-expression of double genes could greatly increase the host strain on ethanol tolerance.

Key words:Keywords: Bacillus pseudofirmus OF4，alcohol dehydrogenase，aldehyde dehydrogenase，ethanol tolerance，co-expression