Site-specific Mutagenesis And Catalytic Site Identification Of Yeast Aryl Alcohol Dehydrogenase In Chinese Rice Wine

In the fermentation process of Chinese rice wine, especially amino acid metabolism of S-accharomyces cerevisiae in Ehrlich pathway,in which Saccharomyces cerevisiaes participate.Phenylalanine and other five kinds of aromatic, aliphatic amino acid was metabolismed to fusel 2-Benzene ethanol respectively by Ehrlich pathway. Which play a decisive role in the yeast fermented food and beverage flavor composition. Catalyzing alpha-aldehydes and ketones to the corresponding alcohols is the last step by the key enzyme in the Ehrlich pathway. Studies show that the seven prediction of aromatic alcohol dehydrogenase of Saccharomyces cerevisiae (AAD) Aad3, Aad4, Aad6,Aad10, Aad14, Aad15 and Aad16 are the key candidate genes, which catalyzed of the last step reaction of Ehrlich pathway.Seven aromatic alcohol dehydrogenase of Saccharomyces cerevisiae in the yeast genome,Aad4 and Aad14 can encode active protein, but Aad3, Aad 10, Aad6/Aad16 and Aad15 may have experienciend some evolutionary events in the evolutionary process (mutation), cannot encode active protein. Therefore, through the bioinformatics analysis of Aad3. Aad6, Aad10, we want to explore the presence of activity in the evolution of these enzymes. Regard the relatively detailed biological characterization of Pc (Phanerochaete Chrysosporium) Aad1 as a reference, through the experiment analysis of molecular informatics on Aad3; Aad10, Aad6, each catalytic site key point was mutatied,cloned and expresed in Escherichia coli, and purified by affinity chromatography with His-tag fusion protein. The fusion protein was purified and tested. According to the analysis of enzyme activity parameters, the key amino acid sites of these enzymes were analyzed. The following conclusions were obtained from the experiment:(1) To determine the key tyrosine sites in Aad protein; (2) In addition to tyrosine Tyr73 sites, there are other mutations involved in the inactivation of Aad3; (3) In the evolutionary process,truntion of the original Aad6 reading frame is an important cause of Aad6 inactivation, and the enzyme parameters analysis of different substrates Aad6p showed different enzyme activity;(4)The process of evolution, the loss of G518 is the cause of Aad10 inactivation. Through these four results, we confirmed the hypothesis:test strains of Saccharomyces cerevisiae AAD family is experienciend the evolution/transformation, the aromatic alcohol dehydrogenase enzyme will als-o provide a powerful engineering to the synthesis of aromatic compounds in Saccharomyces ce-revisiae.