Cloning And Expression Of Alcohol Dehydrogenase And Acetaldehyde Dehydrogenase

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are enzymes which catalyze alcohol and aldehyde with the action of nicotinamide adenine dinucleotide as a coenzyme. They have a considerable research value in application of industry and medicine. In recent years, ADH and ALDH have caused the attention of scholars at home and abroad, because of the development of organic phase catalysis and membrande reactor which play a role in the application of ADH and the widely used of ALDH in its related diseases. These two enzymes generally exist in animals and microorganisms, and now quite a few scholars have studied their separation and purification. However, there are series of problems such as low yield and costly in the extraction of ADH and ALDH from animal liver and microorganisms.In this paper, alcohol dehydrogenase gene and aldehyde dehydrogenase gene were cloned and expressed by means of molecular biology to obtain large quantities of ADH and ALDH, the main results were as follows:1. adh2and aldh6were cloned from yeast by PCR, and connected to pUCm-T vector. Sequence analysis of the gene revealed the similarity is96%and99%respectively compared with the published sequences, and the accession number of this two genes are JX901290and JX901291. 2. adh2was expressed by the expression system of Escherichia coli. The target gene was inserted in the expression vector, and cultivated in37℃. The activity of ADH peaked at0.15U/mg after fermentation under the induction of IPTG.3. adh2and aldh6were expressed by the expression system of Saccharomyces cerevisiae. The ADH activity of the recombinant strain I-ADH could achieve the peak of0.49U/mg, which is2.7times of the original strain. The ALDH activity of the recombinant strain I-ALDH could achieve the peak of0.11U/mg, which is2times of the original strain.4. In order to achieve common expression of two kinds of enzymes in the yeast Saccharomyces cerevisiae INVsl, adh2and aldh6were coupled in the same expression vector pYES2, and the expression vector pYES2-adh2-aldh6was successfully constructed. The highest ADH enzyme activity of the recombinant strain I-ADH-ALDH was0.32U/mg, and the highest ALDH enzyme activity was0.082U/mg.5. An ADH gene was cloned from the genome DNA of Acetobacter pasteurianus, and its similarity was98%compared with JF732899.1in GenBank. The target gene was insert in the pET expression vector. The results of SDS-PAGE shows the target band is appeared as expected and the size of the specific band is consistent. The ADH activity of the recombinant peaked at0.42U/mg after6h fermentation in28℃under the induction of IPTG...