Ju Jiansong, Ma Ning, Zhao Ranran, Liu Jingwei, Xu Shujing, Zhao Baohua
College of Life Science, Hebei Normal University, Shijiazhuang 050024, China.
Wei Sheng Wu Xue Bao. 2013 Apr 4;53(4):363-71.
To clone and characterize the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from Bacillus pseudofirmus OF4.
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.
The optimal temperature and pH of ALDH was 35 degrees C and 8.0, the specific activities of ALDH was 979.6 U/mg protein, the thermostability at 25 degrees C and 35 degrees C was better than at 45 degrees C. 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.
Adh and aldh from B. pseudofirmus OF4 were cloned successfully. Co-expression of double genes could greatly increase the host strain on ethanol tolerance.
克隆并鉴定来自类芽孢杆菌OF4的乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）。
通过PCR克隆adh和aldh基因；构建表达载体pET - Ahd和pET - Aldh并在大肠杆菌BL21（DE3）中表达。经镍 - 氮三乙酸柱层析纯化后，对蛋白质进行鉴定。
ALDH的最适温度和pH分别为35℃和8.0，其比活性为979.6 U/mg蛋白质，在25℃和35℃下的热稳定性优于45℃。虽然ADH的表达水平过低无法纯化，但通过共表达和乙醇耐受性实验发现其具有较高的催化活性。
成功克隆了类芽孢杆菌OF4的adh和aldh。双基因共表达可大大提高宿主菌株的乙醇耐受性。
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