Expression And Characterization ADH2 And ALDH Of Saccharomyces Cerevisiae And Expression And Activity Analysis Of Two Anti-microbial Peptides

1 Expression and Characterization ADH2 and ALDH of Saccharomyces cerevisiaeAlcohol dehydrogenases and Aldhyde dehydrogenases have been emerging as crucial enzymes in ethanol through conversion of acetaldehyde to acetic acid.In this study, ADH2 and ALDHs genes have been cloned and purified as histidine-tagged fusion protein.These enzymes were assaied enzymatic activity in vitro. These data were laid a theoretical foundation for new hangover food. The main contents are illustrated as following:1.1 ADH2 and ALDHs gene have been amplified from genomic of the Saccharomyces cerevisiae.then was inserted into an expression vector to get six recombinant plasmids:pRX2-ADH2,pRX2-ALDH6,pRX2-ALDH2,pRX2-ALDH3,pRX2-ALDH4 and pRX2-ALDH5, respectively. The recombinant plasmid was transformed into Escherichia coli BL21, respectively.And proteins were produced by auto-induction. Six proteins were expressed, but ADH2 and ALDH4 were mainly expressed with inclusion bodies.1.2 ald2 and ald4 genes were constructed the prokaryotic expression vectors of pGEX-ADH2, pBAD-ADH2 and pBAD-ALDH4, Using optimizing erpression condition,these proteins were erpressed with soluble.ADH2 also have mainly expression with inclusion bodies, while ALDH4 have expression with more soluble.1.3 Recombinant proteins with 6His-Tag were purified by Ni-NTA His·Bind Resin. The purity of proteins are nearly 90% analyzed by SDS-PAGE.The concentration of recombinant protein ADH2, ALDH6, ALDH2, ALDH3 and ALDH5 is 2.07 mg/mL,1.624mg/mL,0.6635mg/mL,0.7596 mg/mL and 0.9615 mg/mL determined by Bradford method.1.4 Refolded ADH2 and purified ALDHs were assaied enzymatic activity in vitro. According to the increase in OD340nm for approximately 5 min at 25℃, pH 8.0 of both the test and blank, the specific activity of the purified protein ADH2,ALDH6,ALDH2,ALDH3 and ALDH5 was 2.107 U/mg,16.86 U/mg,1.416 U/mg,1.566U/mg and 2.742U/mg;1.5 ADH2 and ALDHs were analyzed on the optimum temperature, the optimum pH, metal ions and the enzyme kinetic properties.The optimal temperature of ADH2, ALDH6, ALDH2, ALDH3 and ALDH5 was 45℃,30℃,37℃,37℃,50℃, respectively. The optimal pH of the ADH2, ALDH6, ALDH2, ALDH3 and ALDH5 was 7.4,7.4,8.0,7.4,8.5, respectively.1.6 The enzyme kinetic properties of ADH2 and ALDHs were analyzed. The Km for NAD and alcohol of the recombimantly expressed ADH2 were1.1mM and 120μM The Km for NADP and acetaldehyde of the recombimantly expressed ALDH6 and ALDH5 were 101μM,42mM and 664μM,129uM. The Km for NAD and 3-aminopropanal of the recombimantly expressed ALDH2 and ALDH3 were 471μM, 17.5mM and 410μM,8.6μM.2 Expression and Activity Analysis of Two Anti-microbial PeptidesAnti-microbial peptides sequence from two kinds of aquatic animals have been cloned into the prokaryotic expression vector pGEX-4T-1, constructing two fusion protein expression vector pGEX-Y18 and pGEX-CECl,which were transformed into Escherichia coli Rosetta to express,and fusion proteins were expressed mainly in inclusion body. Fusion proteins was renatured,and eleaved to produce antimicrobial peptides Y18 and CEC1. Antimicrobial tests showed that not only fusion protein GST-Y18 and GST-CEC1 but also antimicrobial peptide Y18 and CEC1 can effectively inhibit the growth of Escherichia coli DH5a, Staphyloccocus aureus Rosenbach, Bacillus subtilis, Saccharomyces cerevisiae.The deta was laied a good foundation for antimicrobial peptide genes for subsequent sequence of the single thread highly expressed experimental.