High Activity Of Ethanol / Rational Design Selection And Enzyme Activator Of Aldehyde Dehydrogenase Strain

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are widely distributed in mammalian liver tissues and microorganisms, plant tissues. The commercialized ADH is mostly derived from animal tissues, and the microorganisms as a kind of abundant resource producing ADH can be widely used. A kind of human alcoholic diseases are related to the lack of ALDH activity. Using the computer-aided drug design technology to select and design ALDH activator, to help to improve the enzymatic activity of ALDH is an effective strategy for the treatment of alcohol disorders.In this study, the experimental methods and the following conclusions are the followings:1) ADH/ALDH strain screening, optimization of the fermentation process and determination of fermentation kinetics. Using vinegar grains as plentiful sources of ADH producing strain, we selected a strain Ph-5with acid yields10.8g/L from sugar-free medium. Through the optimization of the medium, determine its optimal fermentation medium is the following:yeast extract (15g/L), glucose (6g/L), anhydrous ethanol (30ml/L); and the optimal culture temperature is37℃. From the growth and acid production curve, we conclude that the strain reach to the stable phase after48h, and the rapid of acid production is the fastest; extraction time of ADH is identified as72h, the time that bacterial yield and ADH specific activity is the highest, besides the ethanol and CaCO3of the medium are the important factors to increase strain ADH activity.2) Study of the Ph-5ADH/ALDH activity. Select the lysozyme method as the best method to extract ADH/ALDH crude enzyme solution. And the ADH refined extraction condition is50%to65%ammonium sulfate saturation, using the extraction method, enzyme activity is increased to3576.35U/mg, and the purification factor is2.7. The optimal pH of the Ph-5ADH is about7.0, while its highest pH stability is at pH7; optimal temperature is75℃, while the ADH enzymatic activity lost only9.90%when it’s preserved at75℃for an hour, proving Ph-5is a kind of heat-resistant enzymes.3) Use the BioSolveIT LeadIT software to dock ALDH protein from the RCSB Protein Data Bank (ID:3INJ) and small molecule ligand from ZINC、Pubchem Compound and TCM Database ligand libraries. The LeadIT score25kinds of small-molecule ligands, by simulating the binding conformation of them and ALDH structure.,4) Enzyme activity experiment to verify the effect of small-molecule ligands on ALDH. The experiment proved4,4’-diaminodiphenylether can act as a kind of ALDH enzyme activator,83μM4,4’-oxybisbenzenamine can increase ALDH enzyme activity by2.25-fold.Through this study, we screened a kind of using ethanol as substrate to produce ADH and ALDH strains; fermentation kinetics and ADH enzyme activity enrichs ADH basic theoretical research. Besides, with the help of computer-aided drug design technology, we determine a new ALDH enzyme activator4,4’-oxybisbenzenamine, in order to improve the enzymatic activity of ALDH.