乙醛脱氢酶的克隆表达及其酶学性质

Expression and enzymatic properties of acetaldehyde dehydrogenase

将Gene Bank中人源乙醛脱氢酶2的基因序列根据酿酒酵母(Saccharomyces cerevisiae)的密码子偏好性进行密码子优化后合成目的基因ALDH2,采用融合PCR技术,构建人源ALDH2基因的表达组件TRP1L-URA3-TPIp-ALDH2-TPIt-TRP1R。通过电转化的方法将基因表达组件通过同源重组整合到酿酒酵母W303-1A的基因组上,获得基因工程菌W303-ALDH2。工程菌发酵后的粗酶液经His TrapTMexcel亲和层析柱纯化获得重组ALDH2,其活性为20.70 U/mg;重组酶的相对分子质量是56 k Da;最适反应pH和温度分别为5.0和35℃;除Na+外,K+、Ca2+、Mg2+、Mn2+均能不同程度地提高ALDH2的酶活。以乙醛为底物测得酶的Km值为0.908 mmol/L,最大反应速度Vmax为114.94 U/mg;以辅酶NAD+为底物测得酶的Km值为0.282 mmol/L,最大反应速度Vmax为58.82 U/mg。

The target gene ALDH2 was synthesized according to the sequences of human acetaldehyde dehydrogenase 2 in Gene Bank after codon optimization of Saccharomyces cerevisiae. Then the ALDH2 gene expression cassette TRP1 L-URA3-TPIp-ALTH2-TPIt-TRP1 R was constructed using fusion PCR and electro-transformed into S. cerevisiae W303-1 A to produce the modified strain W303-ALDH2. The recombinant ALDH2 was obtained after purification by His TrapTMexcel affinity chromatography,and its molecular weight and activity was 56 k Da and 20. 70 U/mg,respectively. The results showed that the optimal reaction pH value and temperature for recombinant ALDH2 were 5. 0 and35 ℃,respectively. Except Na＋,the K＋,Ca2 ＋,Mg2 ＋,Mn2 ＋could increase the activity of ALDH2 in different degrees. When acetaldehyde was used as substrate,the Kmvalue and maximum reaction rate Vmaxof recombinant ALDH2 was 0. 908 mmol/L and 114. 94 U/mg,respectively. When NAD＋was used as substrate,the Kmvalue and maximum reaction rate Vmaxof recombinant ALDH2 was 0. 282 mmol/L and 58. 82 U/mg,respectively.